Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
THE USE OF PROTEASOME INHIBITORS FOR TREATING
CANCER, INFLAMMATION, AUTOIMMUNE DISEASE, GRAFT REJECTION AND
SEPTIC SHOCK
FIELD OF THE INVENTION
The present invention relates to the use of proteasome inhibitors for
targetting different cellular functions implicated in cancer, inflammation,
autoimmune
disease, graft rejection and septic shock.
BACKGROUND OF THE INVENTION
The proteasome is a large protease complex. It is the main
nonlysosomal proteolytic system in the cell, and resides in the cytoplasm as
well as in the
nucleus (Jentsch et al., 1995, Cell 82:881). The proteasome possesses up to
five different
peptidase activities, in different catalytic domains (Ciechanover, 1994, Cell
79:13;
Orlowski et al., 1993, Biochemistry 32:1563), and the best characterized ones
are
chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing
(PGPH)
activities (Orlowski et al., 1981, Biochem & Biophys. Res. Com. 101:814; Wilk
et al.,
1983, J. Neurochem 40:842).
The proteasome is regarded as a housekeeping enzyme and a "garbage
collector" to dispose spent proteins. In fact, the proteasome is responsible
for the
degradation of 70-90% of cellular proteins (Rock et al., 1994, Cell 78:761).
Yet its activity
is well controlled and only those destined to be destroyed are timely digested
by the
proteasome. Through recent studies by the applicants (Wang et al., 1998, J.
Immunol.
160:788) and other researchers (Deshaies et al., 1995, EMBO J. 14:303; Yaglom
et al.,
1995, Mol. & Cell. Biol. 15:731; Seufert et al., 1995, Nature 373:78;
Scheffner et al.,
1993, Cell 75:495; Pagano et al., 1995, Science 269:682; Palombella et al.,
1994, Cell
78:773; Cui et al., 1997, PNAS 94:7515; Treier et al., 1994, Cell 78:787; Lin
et al., 1998,
Cell 92:819), it becomes increasingly clear that the proteasome plays critical
and active
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roles in regulating many different cellular functions. This is achieved by
proteasome's
ability to timely, selectively, and irreversibly destroy regulatory protein
factors, and by its
ability to process precursors of regulatory factors into active ones. For
example, the
degradation of several important regulators of cell proliferation such as
cyclin 2, cyclin 3,
cyclin B, p53 and p27K'pl are mediated by the proteasome (Deshaies et al.,
1995, supra;
Yaglom et al., 1995, supra; Salama et al., 1994, Mol. & Cell. Biol. 14:7953;
Seufert et al.,
1995; supra; Scheffner et al., 1993, supra; Pagano et al., 1995, supra). The
activities of
several important regulators involved in cell activation are also controlled
by the
proteasome. For example, IKBa (Palombella et al., 1994, supra), IKB(3 (Cui et
al., 1997,
supra) and c-Jun protein (Treier et al., 1994, supra) are degraded via the
proteasome
pathway; the p50 component of a transacting nuclear factor NF-KB matures after
cotranslational processing of its precursor peptide by the proteasome (Lin et
al., 1998,
supra).
According to sedimentation rates, the proteasome could be purified as
26S and 20S complexes. The 20S proteasome is a cylindrical proteolytic core
composed of
multiple a and ~3 subunits. Each subunit is coded by a different gene in high
eukaryotic
cells and the total number of subunits varies among different species
(Groettrup et al.,
1996, Immunol. Today 17:429). In vitro, the purified 24S proteasomes can
digest small
peptides in an ATP-independent fashion, but they are inactive on intact folded
proteins
(Peters, 1994, Trends in Biochem. Sci. 19:377). The 20S proteasome can bind at
its ends a
19S regulator and forms the 265 proteasome, which degrades ubiquitinated
protein in an
ATP-dependent fashion (Jentsch et al., 1995, supra). The 20S proteasome can
also
complex with an 11 S activator called PA28 (Groettrup et al., 1996, supra) and
form a so-
called immunoproteasome (Realini et al., 1994, J. Biol. Chem. _269:20727),
which is
essential in processing antigenic peptides for presentation by the MHC class I
complex.
PA28 is a ring-like hexamer or heptamer composed of a and (3 subunits (PA28a
and
PA28 ~), both of which are about 29KD in size (Realini et al., 1994, supra;
Ahn et al.,
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1995, FEBS Letters 366:37). It is not clear whether the 20S proteasome can
associate both
the 19S and 11 S regulators at the same time.
There are two better characterized mechanisms regulating the protein
degradation via the proteasome pathway. The first is that of the substrate
selection. This
process is controlled by a cascade of enzymes called the ubiquitin-activating
enzyme (E1),
the ubiquitin- conjugating enzyme (E2) and the ubiquitin ligase (E3) (Jentsch
et al., 1995,
supra). In addition, the 19S regulator controls the entry of the ubiquitinated
protein into the
20S catalytic core. The second mechanism is the activity of the 20S
proteasome, which is
enhanced by the 11 S PA28 (Realini et al., 1994, supra). It is not clear
whether and how the
11 S PA28 exerts its effect on the 26S proteasome, since it and the 19S
regulator do not
seem to associate with the 20S at the same time. Moreover, whether the 20S
complex
exists in parallel to the 26S complex in vivo is still an open question.
Nevertheless, it has
been shown that overexpression of PA28a could indeed augment significantly
antigen
processing by the proteasome in vivo (Groettrup et al.; 1996, supra). Other
controlling
mechanism might also exist. For example, a CDK inhibitor p27k'pi needs to
associate with
Jab-1 in order to translocate into the cytoplasm, where it is degraded through
the
proteasome pathway (Tomoda et al., 1999, Nature 398:160).
Certain peptide aldehydes such as N-acetyl-L-leucinyl-L-leucinal-L-
norleucinal (LLnL) and N-carbobenzyoxyl-L-leucinyl-L-leucinyl-L-norvalinal
(MG115)
are competitive inhibitors of chymotrypsin (Vinitsky et al., 1992, Biochem.
31:9421;
Tsubuki et al., 1993, Biochem & Biophys. Res. Com. 196:1195). These agents
could
effectively block the chymotrypsin-like activity, and to a lesser extent, the
trypsin-like and
PGPH activities of the proteasome (Rock et al., 1994, supra). They have been
employed to
study the function of the proteasome in various cellular processes. A caveat
of such studies
is that these peptide aldehydes are not specific to the proteasome peptidases,
and other
cellular cysteine proteases such as calpain and cathepsin B (Rock et al.,
1994, supra;
Sasaki et al., 1990, J. Enzyme Inhib. 3:195) are also potently inhibited. This
makes some
interpretations less assuring.
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Orlowski et al. in US patent 5,580,854 teach the use of peptidyl
aldehydes and their analogues to inhibit proteolysis mediated by the
multicatalytic
proteinases complex (MPC) or proteasome. The use of such compounds is to
inhibit
intracellular protein degradation, mitosis and proliferation of dividing cell
population. This
reference does not teach any apoptotic effect of proteasome inhibitors.
Palombella et al. in WO 95/25533 teach a method for reducing the
cellular content and activity of NF-kB, a transcriptional factor playing a
central role in
immune and inflammatory response, by using proteasome inhibitors, peptidyl
aldehydes.
Stein et al. in WO 95/24914 teach a method for reducing the rate of
intracellular protein breakdown by inhibiting proteasome activity. The
inhibitor MG 101
given as an example is shown to be an inhibitor of 26S proteasome. This
inhibitory effect
may result in inhibiting destruction of muscle proteins, antigen presentation
and
degradation of p53 .
Omura et al. have reported in 1991 the discovery of lactacystin (LAC)
which could induce a neurite outgrowth (Omura et al., 1991, J. Antibiot.
44:113; Ibid.,
44:117).
Fenteany et al. have subsequently found that LAC is a
proteasome-specific protease inhibitor (Fenteany et al., 1995, Science
268:726). It inhibits
the three major peptidase activities (i.e., chymotrypsin-like, trypsin-like,
and PGPH
activities) of the proteasome, and the inhibition of the first two is
irreversible in in vitro
assays. LAC does not affect other proteases such as calpain, cathepsin B,
chymotrypsin,
trypsin, and papain.
Schreiber in WO 96/32105 teaches lactacystin and various analogs to
treat conditions that are mediated by the proteolytic function of the
proteasome such as
2f rapid elimination and post-translational processing of proteins involved in
cellular
regulation, intercellular communication and immune response, specifically
antigen
presentation.
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Griscavage et al. (1996, PNAS 93:3308) teach that proteasome activity
is essential for the induction of nitric oxide synthase and that the
proteasome peptidyl
aldehyde inhibitors inhibit the induction of nitric oxide synthase. Nitric
oxide production
is implicated in initiating and exacerbating symptoms of acute and chronic
inflammation
(Lundberg et al., 1997, Nature Medecine 3:30-31). Thus the proteasome
inhibitors,
peptidyl aldehyde, by inhibiting nitric oxide induction have an anti-
inflammatory activity.
There is no teaching of reproducing the same effect using LAC which is more
specific to
proteasome than peptidyl aldehydes.
Cui et al. (1997, supra) had shown that T-cell hybridoma can be
activated using dishes coated with anti-CD3. Once activated these cells die of
apoptosis. It
was demonstrated that lactacystin is an inhibitor of activation induced cell
death (AICD)
and, in these activated hybridoma T-cells, lactacystin must be administered
within 2 hours
of activation to efficiently block AICD. The same authors state that at higher
doses LAC
induces apoptosis in the artificial hybridoma T cells.
Grimm et al. (1996, EMBO 15:3835-3844) have shown that
proteasome plays a role in thymocyte apoptosis and that peptidyl aldehyde
derivatives that
inhibit proteasome and LAC block apoptosis in some cases. In addition Grimm et
al.
(supra) reported that the LAC block of apoptosis was irreversible even when
the drug was
removed from the cell media. Imajoh-Ohmi et al. (1995, Bioch. Biophys. Res.
Com.,
217:1070-1077), teach that lactacystin induces apoptosis in human monoblast
U937 cells.
The involvement of mitochondria in the apoptotic process has been
described by Kroemer et al. (1997, Immunology Today, 18:44). Teachings
relating to the
mitochondria) control of apoptosis at the induction phase that appear to be
essential are
provided.
None of these references teach that proteasome inhibitors eliminate
activated normal cells. There is no teachings in these references of the
involvement of
proteasome activity in mitochondria) function. In addition, these references
do not describe
in mammalian cells what proportion of the protease activity is derived from
the
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proteasome and whether there are efficient and simple methods to screen for
additional
proteasome inhibitors.
LAC is a specific inhibitor of proteasome, but it is mildly toxic and
unstable in aqueous solutions of high pH. LAC and some of its analogues binds
directly to
the proteasome and inhibits three peptidase activities of the proteasome.
However, cellular
events downstream of the proteasome are not totally clear. Knowledge of these
down
stream events related to proteasome activity will allow development of
strategies and
compounds capable of complementing, synergizing, or substituting the effect of
proteasome inhibitors to maximize their effects and/or to minimize their side-
effects.
DPBA is also a potent proteasome inhibitor, competitively inhibiting its
chymotrypsin-like activity (Palombella et al., 1998, PNAS 95:15671; Adams et
al., US
patent 5,780,454). It has a long half life in aqueous solution (T=i2 30 days)
and Dr.
Grisham has shown that, in vivo, DPBA can effectively inhibit Streptococcus
cell wall-
induced polyarthritis in rats without apparent toxicity (Palombella et al.,
1998, supra).
It therefore appears that there is a need to investigate the role of
proteasome, namely that of LAC and DPBA and their analogues in the different
cellular
processes discussed above, and to develop an efficient screening method for
searching
additional proteasome inhibitors.
The present invention seeks to meet these and other needs.
The present description refers to a number of documents, the contents
of which are herein incorporated by reference.
SUMMARY OF THE INVENTION
Applicants have revealed that PA28 a and ~ expression is upregulated
during T cell activation, and probably as a result, the ex vivo proteasome
activity is
fourfold higher in the activated T cells than that in the resting T cells
(Wang et al., 1996,
Eur. J. Immunol. 27:2781 ). Such an augmented activity likely reflects the
increased need
to destroy short-lived regulatory proteins and other types of proteins during
T cell
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activation and proliferation. Consequently, it is logical to hypothesize that
blocking the
proteasome activity will interfere with the activation and proliferation of T
cells.
The applicants are the first ones to have documented critical roles of the
proteasome in lymphocyte activation and proliferation. They have shown that
LAC
strongly inhibits mitogen-stimulated T cell proliferation when the compound is
added
anywhere between the GO and late Gl phase. This indicates that the proteasome
activity is
required from the early until the late G 1 phase for a successful S phase
entry.
Mechanistically, the applicants have shown that activation of CDK2 and cyclin
E-
associated CDK2, which is pivotal for the S phase entry, is proteasome-
dependent.
Furthermore, it is demonstrated that degradation of a Gl phase CDK inhibitor
p27k'pl is
blocked by LAC. This is a likely mechanism for the inhibition of cyclin E-
associated
CDK2 by LAC. Additional results have shown that the proteasome inhibition
supresses
upregulation of p21°'pl and CD25 in early G1 phase. These two events
are also important
for full T cell activation and proliferation (pepper et al., 1983, J. Immunol.
131:690;
Labaer et al., 1997, Genes & Development 11:847). Applicants emphasize that
the
proteasome might also control other cellular events essential for T cell
proliferation. In
any case, the conclusion of these in vitro results are that proteasome
inhibitors can
effectively inhibit T cell activation and proliferation. This suggests that
such inhibitors
can be used as immunosuppressants in the induction phase of organ
transplantation.
The invention demonstrates that proteasome is essential for progression
of T cells from Go to S phase. Taking advantage of LAC's specificity and
potency, this
compound was used to investigate the role of proteasomes in T lymphocyte
activation and
proliferation. It is demonstrated that the proteasome is essential for
progression of T cells
from the Go to S phase. Probably as a result of blockage of cycling, the
activated but not
resting T cells underwent apoptosis when treated with LAC. It is also shown
that the
proteasome controls the protein level of p21 C'p 1 and p27K'p 1 as well as the
CDK2 activity
in the G1 phase, and such control mechanism might be essential in the cell
cycle
progression. LAC can effectively inhibit T cell proliferation even if added at
the Gl/S
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boundary. This knowledge is useful in administering LAC to reverse ongoing
graft
rejection during the rejection episode.
In addition to inhibiting T cell proliferation, proteasome inhibition
causes death of activated but not resting T cells. Applicants are the first to
demonstrate
this phenomenon. It is shown that LAC can induce apoptosis in cycling Jurkat
cells and in
mitogen-activated T cells, but not much in resting T cells. Additional
mechanistic study by
Applicants showed that proteasome inhibition results in reduced degradation of
a pro-
apoptic Bcl-2 family member, and the accumulation of Bik contributes the LAC-
induced
apoptosis. Applicants' results suggest that by inhibiting the proteasome
activity, it is
possible to clonally delete activated alloantigen-specific T cells in vivo,
and achieve long-
term graft tolerance.
Thus the present invention relates to inducing apoptosis of activated
T cells and T cell leukemia but not resting T cells with LAC or its analogues.
Elimination
of malignant cells by a proteasome inhibitor-induced apoptosis is useful in
cancer therapy.
In addition, normal T cells that become activated can be induced to undergo
apoptosis with
a proteasome inhibitor thus eliminating antigen specific T cells. This is
useful in
ameliorating autoimmune diseases and graft rejection by generating antigen
specific
tolerance.
The invention further uses the knowledge of the proteasome
involvement in protein degradation and in the steps for the induction of
nitric oxide
synthase and the effect of LAC or its analogues on the expression of nitric
oxide synthase
and the production of nitric acid. This is useful in the prevention of septic
shock and as an
anti-inflammatory.
The present invention also relates to the inhibition of proteasome
activity by LAC or its analogues such that the inhibition interferes with cell-
cell
interaction during lymphocyte activation in mammals and the up-regulation of
the
adhesion molecule ICAM-1 is repressed. This is useful to control undesirable
immune
responses during graft rejection, autoimmune diseases and inflammation.
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The applicant is the first to show that the electron transport chain in
mitochondria is dependent on the intact activity of the proteasome. The
addition of
proteasome - specific inhibitor such as LAC reduces the electron transport at
the complex
IV of the respiratory chain. The addition of exogenous cytochrome C reverses
this effect.
The effect of LAC on mitochondria has potential applications for disorders
that relate
directly or indirectly to increased activity of mitochondrial function. As
well, since
proliferating cells have a higher energy requirement, inhibition of
mitochondrial
respiration could effectively curb the proliferation of cancer cells and
activated T cells by
depriving the cells of energy, with minimal detriment to normal resting cells.
The applicant is further providing a method for screening proteasome
inhibitors by assaying cellular proteinases activity with a tagged peptide
substrate. It is
understood that this assay protocol can be used in a large through-put
screening procedure
and that any means of tagging peptide substrates specific to different
protease activities of
the proteasome and any means for detection known to a person skilled in the
art; can be
used and incorporated into the large through-put procedure. All the elements
comprising a
method for screening proteasome inhibitors can be incorporated into a kit.
Applicants axe the first to show the dual role of the proteasome in
lymphocyte proliferation and apoptosis, which indicates that proteasome
inhibitors will be
useful irrimunosuppressants in treating allograft rejection in
transplantation. Applicants
tested this hypothesis in a mouse heart transplantation model. Since DPBA is
more stable
than LAC in aqueous solution (Palombella et al., 1998, supra), the Applicants
chose the
former for this in vivo study.
Therefore, in accordance with the present invention it is provided:
The use of a proteasome inhibitor to induce apoptosis in proliferating
cells, wherein said proteasome inhibitor may be lactacystin or an analogue
thereof and said
proliferating cells are cancerous cells and/or activated T cells, such that
activated T cells
are antigen induced. The above cells are stopped from progressing from Go to
G1/M in a
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cell cycle as a consequence of proteasome inhibition. As well, CDK2 and the
associated
Cyclin E activities are substantially inhibited, whereby said cell cycle
progression is
substantially arrested. Additionally, CDK4 cell activity is not inhibited.
Any one of the use of the above stated provisioned uses of a
proteasome inhibitor, wherein said proliferating cells are eliminated and
cancer
progression is arrested and, activated T cells are eliminated.
The use of a proteasome inhibitor to reverse graft rejection in a patient
in need for such a treatment comprising the step of administering to said
patient an
apoptotic amount of a proteasome inhibitor when said patient T cells are
activated wherein
said patient is in need of said treatment when an ongoing allograft rejection
occurs or at
least 24h after graft transplantation.
The use of a proteasome inhibitor in the making of a medicament to
induce apoptosis in proliferating cells. The use of a proteasome inhibitor as
defined in the
above stated provisions, alone or in combination with another medication, to
eliminate or
to reduce antigen-specific induced T or B cells; and achieve antigen-specific
tolerant status
or reduced responsiveness to an antigen in a patient which condition requires
such
treatment wherein said condition is selected from the group consisting of:
autoirnmune
disease, graft rejection and inflammation.
A method for screening a compound for proteasome inhibition activity,
which comprises: obtaining a mammalian cell lysate comprising proteasomes, a
partially
purified proteasomes preparation or a purified proteasomes preparation;
tagging at least
one peptide substrate specific to a known proteasome protease activity;
combining said
proteasomes and said at least one tagged peptide substrate; contacting the so
combined
proteasomes/tagged peptide substrate with said compound; said at least one
tagged peptide
substrate fails to release tag if said compound is a proteasome inhibitor, and
detecting a
decrease or absence of the released tag in the presence of said compound
relating to the
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released tag in the absence of said compound as an indication of proteasome
inhibition
activity for said compound wherein said at least one tagged peptide substrate
is a
fluorogenic peptide and wherein said proteasome protease activity is trypsin-
like
chymotrypsin-like or peptidylglutamyl-peptide hydrolyzing activity.
The use of a proteasome inhibitor to disrupt mitochondria) function,
wherein said inhibitor blocks electron transport in said mitochondria and,
wherein said
inhibitor blocks said electron transport at complex IV in said mitochondria
such that
mitochondria) function is disrupted, W herein disruption of mitochondria)
function is
corrected by cytochrome C. The use of the afore-mentioned provisions relating
to
mitochondria) function to treat a pathological condition wherein high
mitochondria)
activity occurs, said pathological condition is selected from the group
consisting of:
cancer, inflammation, undesirable immune responses and hyperthyroidism.
The use of a proteasome inhibitor to disrupt nitric oxide synthesis,
wherein the proteasome inhibitor inhibits nitric oxide synthase gene
expression.
An apoptotic composition comprising a therapeutically effective
amount of a proteasome inhibitor and a pharmaceutically acceptable carrier
which may
additionally comprise a therapeutically effective amount of an inhibitor to
CDK4 activity
and/or a therapeutically effective amount of an inhibitor to CDK2 activity and
more
particularly to Cyclin E activity, a therapeutically effective amount of an
inhibitor which
prevents p2lC'pl upregulation blocks the degradation of p27k'p~ and a
therapeutically
effective amount of an inhibitor which prevents CD25 upregulation.
The use of cyclosporin A, rapamycin or FK506 as a proteasome
inhibitor.
A composition for use in inhibiting graft rejection comprising a
therapeutically effective amount of cyclosporin A, rapamycin or FK506 in
combination
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with a therapeutically effective amount of a proteasome inhibitor and may be
in
combination with a therapeutically effective amount of an inhibitor of ICAM-1
expression.
A composition for use in inhibiting graft rejection comprising a
therapeutically effective amount of an inhibitor which suppresses expression
ICAM-1 in
combination with a therapeutically effective amount of a proteasome inhibitor.
The use of a proteasome inhibitor to alleviate a disease or a disorder,
wherein adhesion molecule ICAM-1 is upregulated and said disease or a disorder
is graft
rejection, autoimmune disease or inflammation.
The use of a proteasome inhibitor is to alleviate a desease or a disorder
wherein at least one of CDK2, p21~'pl, CD25 is upregulated and/or p27k'pl
degraded,
wherein said disease or disorder is graft rejection, autoimmune disease or
cancer.
The use of a proteasome inhibitor to alleviate a disease or disorder,
wherein nitric oxide synthase is upregulated and said disease or disorder is
inflammation
or septic shock.
The said proteasome inhibitor may be used alone or in combination
with any drugs known in the art for use in treating cancer, inflammation,
autoimmune
disease, septic shock or inflammation.
The use of all the afore-mentioned provisions wherein said proteasome
inhibitor is particularly lactacystin or DPBA or their analogues thereof, is
within the scope
of this invention. The term "proteasome inhibitor" intends to cover all
molecules having
the capacity to inhibit the proteasomal enzyme activities. Inhibitors are
disclosed in
Vinitsky et al., 1992, supra; Tsubuki et al., 1993, supra and Orlowski et al.,
US Patent
5,580,854. The preferred inhibitors comprises lactacystin and its analogs;
examples of
such analogs are disclosed in Omura et al., 1991, 44:113; Ibid., 44:117 and in
Schreiber,
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WO 96/32105. The preferred inhibitors also comprise dipeptide boronic acid
(DPBA) and
its analogs; examples of such analogs are described in US 5,462,964, US
6,083,903 and in
US 5,780,454.
BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the invention, reference will now be
made to the accompanying drawings, showing by way of illustration a preferred
embodiment thereof, and in which:
Figure 1 shows that LAC strongly inhibits T and B cell proliferation.
Lymphocytes were stimulated with various mitogens as indicated, and LAC at
different
concentrations was added at the beginning of the cultures. The cells were
pulsed with
3H-thymidine between 48h and 64h. Samples were in triplicates. All the
experiments were
performed at least three times and similar results were obtained.
Representative results are
shown.
A: Peripheral blood T cells stimulated with PHA (2 ~g/ml).
B: Peripheral blood T cells stimulated with OKT3 (SOng/ml).
C: Peripheral blood T cells stimulated with anti-CD28 (SOng/ml) plus ionomycin
(1 ~ag/ml).
D: Tonsillar B cells stimulated with SAC (1:15,000 dilution) and IL-2 (100
~/ml).
Figure 2 shows that inhibition of the proteasome activity results in
induction of apoptosis of activated normal cells and leukemic T cells but not
resting
normal T cells. Tonsillar T cells (A, B, and D) and Jurkat cells (C and E)
were treated with
LAC (10 uM for T cells and 6 uM for Jurkat cells). LAC was added at the
beginning of
the culture or 40h after T cell activation as indicated. The cells were
harvested at the time
points as shown. They were evaluated for their viability with trypan blue
exclusion (A, B,
and C), and for their mode of cell death according to DNA fragmentation (D and
E).
Figure 3 shows by electron microscopy that the proteasome inhibitor
induced apoptosis in activated T cells and Jurkat cells.
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A and B: Morphology of resting T cells treated with LAC. Tonsillar T cells
were
culture in the absence (A) or presence (B) of LAC (lOmM) for 24h, and the
cells were
examined by EM.
C and D: Morphology of activated T cells treated with LAC. Tonsillar T cells
were
first activated with PHA (2 ug/ml) for 40h. The cells were then cultured in
the absence (C)
or presence (D) of LAC (10 uM) for additional 24h, and were examined with EM.
E and F: Morphology of Jurkat cell treated with LAC. Jurkat cells were
cultured in
the absence (E) or presence (F) of LAC (6 uM) for 24h and were evaluated with
EM.
Arrows indicate condensed nuclei.
Figure 4 shows that the effect of LAC is rapid and reversible in cell
culture.
A. The rapid effect of LAC Peripheral blood T cells were pretreated with 10 uM
LAC in culture medium or in culture medium alone for 3h or 16h. The cells were
then
washed and recultured in the presence of 2 ug/ml PHA for 64h. The cells were
pulsed with
3H-thymidine for 16h before they were harvested at 64h. Samples were in
triplicates.
B. The inhibitory effect of LAC on the proteasome activity was reversible in
the
cells Jurkat cells were pretreated with LAC (6 pM) in culture medium for 3h.
The cells
were washed and recultured at 0.5 x 1 O6 cells/ml for Oh, Sh or 21 h. The
cells were then
harvested, washed and sonicated. The lysate protein (20 ug/sample) was assayed
for its
proteinase activity under a condition at which 90% of the activity was
attributed to the
proteasome. The samples were in duplicates. The result is expressed as
relative
fluorescence intensity at 440nm.
C. The activity of LAC in culture supernatants is short-lived LAC (6 ~aM) was
added to Jurkat cell culture (0.5 x 106 cells/ml). The supernatants were
harvested at 4h, 6h,
16h and 24h. These conditioned media were used
to culture fresh Jurkat cells for 3h. The cells were then harvested and
assayed for the
proteasome activity as described in Fig. 4B. Samples were in duplicates.
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All the experiments were performed at least three times, and similar results
were
obtained. Representative data are shown.
Figure 5 shows that LAC inhibits CD25 upregulation during T cell
activation.
Peripheral blood T cells were stimulated with PHA (2 ug/ml) for 48h in the
presence or absence of LAC (10 ~zM, added at the beginning of the culture).
CD25
expression on T cells was evaluated by anti-CD25-PE/anti-CD3-FITC two-color
flow
cytometry. Similar results were obtained in two independent experiments, and a
representative one is shown. The data are presented as two color histograms in
forms of
contours, as well as in an overlay histogram.
Figure 6 shows the role of the proteasome in cell cycle progress.
A. LAC does not inhibit the progress from the G2/M phase to the Gl phase in
synchronized Jurkat cells Jurkat cells were synchronized at the G2 !M phase by
16h
nocodazole treatment. For the last 3h of the treatment, LAC (6 ~M) was added
to the
cultures destined to be treated by LAC later. The cells were then released by
washing out
nocodazole, and recultured in complete medium with or without 6 uM LAC. The
cells
were sampled at Oh, 4h and 8h after the GZ/M release, stained with propidium
iodide, and
analyzed with flow cytometry.
B. LAC slows the cell cycle progress from the Gl/S boundary to the G2lM phase
in
synchronized Jurkat cells Jurkat cells were synchronized at the Gl/S by
isoleucine
starvation followed by a hydroxyurea treatment. The synchronized cells were
released by
washing out hydroxyurea and were cultured in complete medium in the absence or
presence of LAC (6 uM). The cells were sampled at Oh, 3h, 6h, 9h, 12h, 15h and
24h after
the release, and were stained with propidium iodide and analyzed with flow
cytometry.
C and D. LAC blocks the S phase entry of the mitogen-stimulated peripheral
blood
T cells Peripheral blood T cells were stimulated with PHA (2 u/ml) in the
absence or
presence of LAC (10 uM, added at Oh, 16h, 24h, or 40h, as indicated in the
bottom of the
panels). For the flow cytometry analysis of the cell cycle progress, the cells
were harvested
CA 02372316 2002-02-21
-16-
at Oh, 16h, 40h and 64h as indicated on the top of the panels (Fig. 6C). For
3H-thymidine
uptake, the triplicated cell samples were pulsed at 48h and harvested at 64h
(Fig. 6D)
The experiments were performed three times, and similar results were obtained.
Representative data are shown.
Figure 7 shows the results of the kinase assays for the effect of LAC on
CDK activity.
Tonsillar T cells were activated with PHA (2 pg/ml) for a period as indicated
in
each graph. LAC (10 uM) was added once at Oh. The cells were harvested at 16h,
24h, or
40h as indicated. An equal amount of lysate protein (40 /sample) was
precipitated with
rabbit anti-CDK4, anti-CDK2 or anti-Cyclin E antisera (2.5 ug Ab/sample). The
immune
complexes were assayed for their kinase activities using histone H1 as a
substrate. (A)
CDK4 kinase activity. (B) CDK2 kinase activity. (C) Cyclin E-associated CDK
activity.
The membrane in (C) was subsequently hybridized with anti-Cyclin E (1 ug/ml)
followed
by 125I-protein A for the evaluation of the protein level of Cyclin E.
All the experiments 'were performed three times, and similar results were
obtained.
Representative data are shown.
Figure 8 shows the results of immunoblotting analysis of the effect of
LAC on the protein levels of Cyclin E and Cyclin A.
Tonsillar T cells were stimulated with PHA (2 ~ag/ml) for 40h in the presence
of
hydroxyurea (1mM), and these cells were blocked at the G1/S boundary (G1
block). The
synchronization was released by washing out hydroxyurea, and the cells were
recultured in
medium containing 2 ~g/ml PHA in the absence or presence of LAC (lOnM, added
once
at the time of the release). The cells were harvested at 6h and 22h post the
Gl/S block. The
cell lysates (40 ~zg/sample) were resolved in 10% SDS-PAGE, and transferred to
PVDF
membranes. The membranes were hybridized with rabbit-anti-Cyclin E or
anticyclin A
antisera followed by 12x1-protein A. The Cyclin E level (Fig. 8A) and cyclin A
level
(Fig. 8B) of representative experiments are shown. Similar results were
obtained in a total
of three independent experiments.
CA 02372316 2002-02-21
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Figure 9 shows the results of immunoblotting analysis of the effect of
LAC on the levels of CDK inhibitors p27K'pl and p2lC'pl.
Tonsillar T cells were stimulated with PHA (2 tzg/ml) for 16h, 40 or 64h in
the
absence or presence of LAC (10 uM). For the 16h and 40h culture, LAC was added
once
at Oh. For the 64h culture, LAC was added once at 40h. The cell lysates were
resolved in
10% SDS-PAGE, and blotted onto PVDF membranes. The membranes were hybridized
with rabbit anti-p27K'pl antisera (Fig. 9A) or with anti-p2lC'p' antisera
(Fig. 9B) followed
by iasl-protein A. The experiments were performed three times, and similar
results were
obtained. Representative data is shown.
Figure 10 shows human peripheral blood mononuclear cells that were
cultured in medium (A), 2 ~g/ml PHA (B), or PHA plus 10 uM lactacystin for
24h.
Lactacystin could effectively block the aggregate formation.
Figure 11 shows mouse lymph node cells that were cultured in medium
(A), 2 ug/ml Con A (B), or Con A plus 10 izM lactacystin for 24h. Lactacystin
could
effectively block the aggregate formation.
Figure 12 shows mouse lymph node cells from TCR transgenic mice
named 2C that were cultured in medium (A), 2 ug/ml Con A (B), or Con A plus 10
uM
lactacystin. After 24h and 48h, the cells were examined for ICAM-1 expression
by flow
cytometry, using FITC-anti-ICAM-1/ 1B2-PE. Monoclonal Ab 1B2 recognize a
clonotypic determinant on the TCR of the transgenic T cells which are largely
CD8
positive (>75%). Lactacystin could effectively block the upregulation of ICAM-
1 on those
CD8 positive T cells.
Figure 13 shows mouse peritoneal exudate macrophages that were
stimulated with 2 ug/ml LPS in the presence of lactacystin at different
concentrations.
Nitric oxide production by the macrophages was measured according to the
nitrate
concentrations in the supernatants.
CA 02372316 2002-02-21
-18-
Figure 14 shows mouse peritoneal exudate macrophages that were
stimulated with 2 ug/ml LPS in the presence or absence of lactacystin (10
p.M). Nitric
oxide synthase expression was measured with Northern blot analysis.
Figure 15 shows that Lactacystin blocks electron transport downstream
of Complex I. Respiration of Jurkat cells (JC) or rat kidney mitochondria
(RKM) was
measured by 02 consumption using an oxygen electrode. The function of Complex
I of
digitonin (Dig)-permeated Jurkat cells was blocked by rotenone (Rot), and the
respiration
was resumed by adding succinate (Suc), which provides electrons to Complex II
directly
and thus bypasses Complex I. The maximal respiration was achieved by adding
CCCP
(carbonyl cyanide m-chlorophenylhydrazone), which uncouples the oxidation and
phosphorylation. The respiration could be blocked by antimycin A (Ant), which
inhibits
Complex II. Curves 1 and 6 represent positive controls of rat kidney
mitochondria. Curves
2 and 5 represent normals untreated Jurkat cells. Curves 3 and 4 represent
Jurkat cells
treated with lactacystin (6 ~1VI) for 2h and 4h, respectively.
Figure 16 shows that Lactacystin blocks electron transport at Complex
IV. Complex III in the respiration chain was blocked at Complex III antimycin
(Ant), and
the electron flow was resumed by addiind ascorbate (Asc) and TMPD (tetramethyl-
p-
phenyl-enediamine). The maximal respiration was triggered by CCCP, and was
totally
inhibited by potassium cyanide (KCN).
Figure 17 shows that Cytochrome completely corrects the defect at
Complex IV caused by LAC. The assay system is identical to that described in
Figure 16.
Jurkat cells were treated with LAC for 4h (curve 3). The decoupling reagent
used in this
experiment to achieve maximal respiration is FCCP (carbonylcyanide-p-
trifluoromethoxyphenylhydrazone).
Figure 18 shows that RAPA, FK506, and CsA inhibit PA28 expression
at the mRNA level. Tonsillar T cells (A) and B cells (B) were cultured in the
presence of
various reagents as indicated (PHA, 2 ug/ml, RAPA, 10 nM; FK506, 10 nM, CsA, 1
uM;
SAC, 1:10 000 dilution; Il-2, 25 U/ml. After 6h, 20h or 40h, the cells were
harvested and
CA 02372316 2002-02-21
-19-
total RNA was analyzed by Northern blotting for PA28 ~i expression. The PA28
(3 message
in T cells was also examined by Northern blotting using a similar condition as
for PA28 ~i
(C). The experiments were repeated more than three times, and representative
ones are
shown.
Figure 19 shows that RAPA inhibits PA28 ~3 and PA28a protein in the
activated T cells. (A) An analysis of PA28 ~ protein by immunoblotting is
shown.
Tonsillar T cells were cultured with 2 pg/ml PHA or PHA plus 50 nM RAPA for
24h. The
cells were harvested and lysed. Forty micrograms of cleared lysate protein per
sample was
analyzed by immunoblotting using rabbit anti-PA28 ~ antiserum. (B) An analysis
PA28a
and PA28 ~i protein by confocal immunofluorescence microscopy. Tonsillar T
cells were
cultured with 2 ug/ml PHA or PHA plus 50 nM RAPA for 24h. The cells were
stained
with antisera specific for PA28a and PA28 (3. Thirteen cells were analyzed for
PA28a
protein and twelve cells for PA28 ~i protein in a blind fashion. The mean + SD
of relative
fluorescence intensity per whole cell is presented. Unpaired Student's t-test
was employed
for statistics. The difference between PHA-activated sample and PHA plus RAPA-
treated
samples was highly significant (p = 3.20 x 10'9 for PA28a and p = 5.99 x 10'5
for PA28 (3).
Figure 20 shows that effect of RA.PA on proteasome activity in human
PBMC. Human PBMC were cultured in the absence or presence of 2 ~zglml PHA or
10 nM
RADA for 16h-70h as indicated. The cells were then harvested, and the
chymotrypsin-like
activity of whole cells lysates was assayed in the absence or presence of 20
uM
proteasome inhibitor LAC. The data are presented as arbitrary units of
fluorescence
intensity per 20 ug lysate protein. The experiments were repeated three times
and a
representative one is shown. Samples are in duplicate and the mean t SD is
shown. (A)
Total chymotrypsin-like activity in the lysate of PBMC. (B) Lactacystin-
inhibitable
chyrnotrypsin-like activity in the lysate of 70h PBMC. Nine micrograms of 20S
proteasome were used as positive controls for the inhibitory effect of LAC at
10 uM and
20 uM. LAC was always added to the lysates during the proteinase assay 15 min
before
CA 02372316 2002-02-21
-20-
the addition of the substrate. The solid bars represent the activity in the
presence of LAC.
The net proteasome activities are calculated as the total activity minus the
remaining
activity after the LAC addition.
Figure 21 shows the elimination of an alloantigen-specific response by
a proteasome inhibitor lactacystin. The C57BL/6 spleen cells (H-2b) were
stimulated with
mitomycin c-treated BALB/c spleen cells (H-2d). On day 2 when most of the H-2d-
specific cells were activated, the mixed lymphocyte culture (MLR) was treated
with
lactacystin (LAC, 8uM) for 3 h. After wash, the cells were put back in culture
for
additional 8 days, and then stimulated with either fresh BALB/c or C3H (H-2k)
spleen
cells. In MLR treated by LAC, the C57BL/6 cells failed to respond to the
BALB/c cells,
but respond well to third party C3H (H-2k) cells. The difference is more
pronounced in
day three of the culture.
Figure 22 shows that the LAC-induced DNA fragmentation is inhibited
by a broad spectrum caspase inhibitor zVAD.fmk. Jurkat cells were treated with
LAC (6
uM) in the absence or presence of different concentrations of zVAD.fins (0.4
uM to 33.3
uM) for 6 h. The cells were harvested and their DNA was analyzed by a DNA
fragmentation assay according to DNA laddering.
Figure 23 shows that preventing the degradation of a pro-apoptotic
Bcl-2 family member Bik is a mechanism for the proteasome inhibitor-induced
apoptosis.
Jurkat cells were treated with lactacystin (6 uM) for 5 h (lanes 2 and 4 of
panel A), 4 h
(lane 2 of panel B) or 7h (lane 3 of panel B), lane 1 in panels A and B is
untreated control
samples. The cells were separated into mitochondrial (mito in panel A and
mitochondria in
panel B) and cytosolic (cytosol in panel A) fractions, and the lysate of these
two fractions
analyzed by immunoblotting using goat anti- Bik, and rabbit anti-Bax, Bak and
Bad Ab
(all from Santa Ciuz Biotech, Santa Cruz, CA) followed by enhanced
chemiluminescence
(ECL, kit from Amersham).
CA 02372316 2002-02-21
-21 -
Figure 24 shows that overexpression of an anti-apoptotic Bcl-2 family
member Bcl-xL in a B cell line could protect the cells from apoptosis caused
by
proteasome inhibition. A human B cell line Namalwa was stably transfected with
an anti-
apoptotic Bcl-2 family member Bcl-xL, and its sensitivity to the proteasome
inhibitor-
induced apoptosis tested by the quantitative filter elution assay (Schmitt et
al., 1998, Exp.
Cell Res. 240:107), which detects DNA fragmentation during apoptosis. T'he
wild type
Namalwa and transfected Namalwa cells overexpressing Bcl-xL were pulsed with
14C-
thymidine for 24 h, and then treated with different concentrations of
lactacystin (0.75 uM,
1.5 ~aM, 3 uM, 6 uM and 10 ~M). The cells were harvested at different time
intervals (24-
96 h), and DNA fragmentation measured.
Figure 25 shows that the wild type Namalwa cells have increased Bik
level after treatment with lactacystin and that the Bcl-xL transfected Namalwa
cells have
overexpressed Bcl-xL. Jurkat cells, wild type Namalwa cells and Bcl-xL
transfected
Namalwa cells were treated with medium (lanes 1), staurosporine (0.3 uM, lanes
2) and
lactacystin (6 ~aM, lanes 3) for 6 H. The proteins from the mitochondrial
fraction of these
cells were analyzed by immunoblotting and the amount of Bik, Bcl-xL, Bax, and
Bak
evaluated. The same membranes were used sequentially and probed with different
antibodies against these factors. A nonspecific band recognized by a
monoclonal antibody
against cytochrome oxygenase (COX) was used as control for even sample loading
in the
lanes.
Figure 26 shows the chemical structure of the proteasome inhibitors
dipeptide boronic acid (DPBA; Pyz-Phe-boroLeu; Pyz, 2, 5-pyrazinecarboxylic
acid) and
lactacystin.
Figure 27 shows the inhibition of the 20S protesome activity by the
proteasome inhibitor DPBA. The 20S proteasome was purified from rat liver as
described
in the applicant's previous publication (1996, supra). A fluorogenic peptide
sLLVY-MCA
was used as a chymotrypsin substrate. DPBA of different concentrations was
added into
the reaction mix, and incubated at 37°C for 30 min. The relative
fluorescent intensity,
CA 02372316 2002-02-21
-22-
which reflects the chymotrypsin-like enzymatic activity of the 20S proteasome,
was
measured with a fluorometer using excitation/emission wavelengths of
380nm/440nm.
Figure 28 shows the suppression of anti-CD3-stimulated T cell
proliferation by the proteasome inhibitor DPBA. BALB/c mouse spleen cells were
stimulated with anti-CD3 (clone 2C11, SOng/ml), and DPBA of different
concentrations
was present in the culture. The cells were pulsed with 3H-thymidine at 48h and
harvested
at 64h after the culture.
Figure 29 shows that the proteasome inhibitor DPBA prolongs mouse
heart allograft survival. BALB/c mice (H-2d) were used as heart donors and
C57BL/6 mice
(H-2b) as recipients. Heterotopic heart transplantation was performed on day
0, and a
proteasome inhibitor DPBA was administrated from day 1 to day 16 i.p, daily.
Graup 2
was given 0.65 mg/kg/day; group 3 was given 1.0 mg/kg/day for 4 days, and the
dose was
then reduced to 0.5 mg/kg/day for 12 days. The graft survival days, mean
survival time
(MST) and the p value (unpaired Student's test) compared with the control
group was
presented.
Figure 30 shows that the proteasome inhibitor DPBA is effective in
treating ongoing heart allograft rejection in mice. The experiment was carried
out as
described in Fig. 29, except that DPBA was only administrated between day 3
and 6 for 4
days, when the rejection is ongoing.
Figure 31 shows that the proteasome inhibitor DPBA effectively
prevents mouse islet allograft rejection. C57BL/6 mice were treated with 250
mg/kg
streptozocin and used as islet graft recipients when their blood glucose
reached 20 nM.
Islets from BALB/c mice were isolated after collagenase digestion followed by
Ficoll
gradient separation. The islets were cultured overnight, and transplanted into
the peritoneal
cavity of the diabetic C57BL/6 recipients (500-600 islets/recipient). Twenty-
four hours
after the transplantation, the recipients were given DPBA i.p. at 1 mglkg/day
for 16 days
and then at 0.5 mg/kg twice a week until day 60 post operation. The blood
glucose of the
mice was measured daily and the means + SDs are shown. The isograft controls
are
CA 02372316 2002-02-21
- 23 -
diabetic C57BL/6 mice transplanted with C57BL/6 islets (500-600
isletslrecipient), and
were not treated with DPBA. The allograft controls were diabetic C57BL/6 mice
transplanted with BALBIc islets (500-600 isletslrecipient) without DPBA
treatement. The
mice were sacrificed on day 60, or when their blood glucose reached 20 nM (the
allograft
control group).
Other objects, advantages and features of the present invention will
become more apparent upon reading of the following non-restrictive description
of
preferred embodiments with reference to the accompanying drawings which are
exemplary
and should not be interpreted as limiting the scope of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to proteasome activities in cellular
processes and any inhibitors of proteasome activities.
Proteasome Activity is Obligatory for Activation and Proliferation of T and B
Cells
The role of proteasome in T cell activation and proliferation was first
examined in PBMC, using the proteasome-specific inhibitor LAC. The PBMC were
activated with various stimulants. LAC was added to the cells in the beginning
of the
culture (Oh) along with the stimulants. 3H-thymidine uptake between 48h and
64h of 64h
cultures was used as a parameter for cell proliferation. As shown in Fig. 1,
LAC strongly
and dose-dependently inhibited the T cell proliferation induced by a T cell
mitogen PHA
(Fig. 1A), by crosslinking TCR with anti-CD3~ (Fig. 1B), or by Ca + ionophore
plus
cross-linking of the T cell co-stimulating molecule CD28 (Fig. 1 C). The
T cell-independent B cell proliferation induced with SAC plus IL-2 in
tonsillar B cells was
also potently inhibited by LAC (Fig. 1D). In all the four systems employed,
LAC at 5 uM
could exert near-to-maximal inhibition. The results suggest that LAC's effect
is not
lymphocyte type(T or B cells)-specific nor stimulant-specific. Rather, it
likely affects
certain downstream events governing a more general processes) in lymphocyte
activation
and proliferation.
CA 02372316 2002-02-21
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LAC Causes Apoptosis in Activated but not Resting T Cells
In one embodiment of the present invention a compound is provided
that induces activated and leukemic T cells to undergo apoptosis.
Since LAC has been reported to induce apoptosis in U937 cells (Chen
et al., 1996, J. Immunol. 157:4297), it is crucial to examine whether the LAC-
induced
inhibition of cell proliferation is due cell death; be it apoptosis or
necrosis.
The viability of T cells and Jurkat cells after LAC-treatment was first
evaluated with trypan blue exclusion. Resting T cells (T cells in medium) or
PHA-stimulated T cells were cultured with 10 pM LAC (LAC added at the
beginning of
the culture). As shown in Figure 2A, after 16h culture, the viability of the
cells only had
minor decreases (< 12%) in LAC-treated cells compared with those without LAC
(97% vs
92% for cells in medium, and 94% vs 83% for cells with PHA). After a prolonged
culture
for 64h, the decreases were more prominent although were still less than
27%(97% vs 79%
for cells in medium, and 90% vs 63% for cells with PHA).
There was a tendency that the activated T cells were more susceptible
to LAC than the resting T cells. This became more evident when LAC was added
to
T cells 40h after the PHA activation (Figure 2B). The viability of the
activated T cells
dropped from 94% to 46% after additional 24h culture, although 9h culture did
not change
the viability significantly according to trypan blue exclusion. On the other
hand, the
viability of the resting T cells in medium had only a small decrease (from 98%
of the
control to 87% of the LAC-treated) after 24h of LAC presence.
Why did LAC added at Oh along with PHA cause less cell death
compared with LAC added at 40h post PHA stimulation (Figure 2A vs 2B)? It will
be
demonstrated that LAC is rapidly degraded in the cell culture. After 24h in
culture
medium, LAC lost its activity, and at 40h when the T cells were fully
activated and
become more susceptible, there was no biologically active LAC in the culture.
This could
explain the observed difference in terms of viability between the Oh and 40h
addition of
LAC to the PHA-activated T cells.
CA 02372316 2002-02-21
-25-
The effect of LAC on Jurkat cells was quite similar to that on the
activated T cells. Less than 8h exposure to 6 uM LAC did not induce apparent
Jurkat cell
death, while about 60% of the Jurkat cells were trypan blue positive after 24h
culture with
LAC (Fig. 2C).
We next employed DNA laddering to study the mode of cell death
caused by LAC, and the result of this experiment also reflected the degree of
cell death
after different treatments. As shown in Fig. 2D, resting T cells treated with
10 uM LAC
for 24h had no apparent DNA breakdown (lanes l and 2). This correlated to the
good cell
viability as shown in Fig. 2B. On the other hand, clear DNA ladders could be
observed
from activated T cells (40h post PHA-stimulation) treated with LAC for
additional 9h
(lanes 3 and 4). After 24h of LAC treatment, the ladders became less discrete,
and this
probably reflected further DNA breakdown. For Jurkat cells, DNA fragmentation
could be
detected as early as 6h after the LAC treatment, and after 16h, the
fragmentation became
more prominent (Figure 2E).
Electron microscopy was also employed to examine the mode of cell
death induced by LAC. The resting T cells (cells cultured in medium, figure
3A), activated
T cells (40h after PHA activation, Figure 3C), and Jurkat cells (Figure 3E)
were all healthy
looking. Occasional condensed nuclei were observed in medium cultured T cells
(Figure
3A) and this is not unusual. The resting T cells treated with LAC (10 uM)for
24h were
still healthy (Figure 3B). However, nuclear condensation, which is a hallmark
of
apoptosis, were frequently observed in activated T cells and Jurkat cells
after they were
exposed to LAC (10 uM and 6 uM, respectively) for 24h (Figures 3D and F).
Following conclusions are drawn from the results of this section. 1 )
Resting T cells or T cells in their early activation phase (less that 24h
after
PHA-stimulation) are not sensitive to LAC in terms of cell viability.
Consequently, there
are still a significant percentage of live cells after 64h culture should LAC
be added once
at the beginning. 2) Less than 8-9h of LAC treatment does not affect
significantly viability
of activated T cells (40h post PHA activation) or Jurkat cells, according to
trypan blue
CA 02372316 2002-02-21
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exclusion. 3) Prolonged treatment (24h) of the activated T cells or Jurkat
cells with LAC
causes cell death in the form of apoptosis, although signs of apoptosis could
be detected as
early as 9h in T cells and 6h in Jurkat cells after the LAC treatment.
The data in this section further infer following notions. 1 ) LAC's
differential effect on the viability of resting versus cycling cells suggests
that it is not
simply nonspecific cytotoxicity, but relates to the status of the cell cycle.
2) The cell death
without doubt contributes to but cannot solely account for the observed
inhibition of
proliferation by LAC, since there are still significant percentage ( about
60%) of live cells
at the end of the culture according to trypan blue exclusion. Moreover, we
will elaborate
later that the cell death is a consequence of blockage of cell cycle progress.
3) Admittedly
the trypan blue negative cells includes some early apoptotic cells, as
evidenced by the fact
that DNA laddering could be detected in a largely trypan blue negative
population.
However, it does not necessarily mean that the whole population is dead. We
will later
demonstrate that most Jurkat cells treated with LAC for 6h to 8h could still
progress
normally in cell cycle, in spite that a certain degree of apoptosis could be
detected in these
cells. 4) LAC could be used to study the role of proteasomes in lymphocyte
activation and
proliferation, as long as the compound is applied only once in the beginning
of activation
of the resting T cells and the experimentation is carried out in 24h-40h, or
LAC is present
for less than 8h in the case of cycling cells, since such treatments do not
drastically affect
the viability of the cells.
A specific embodiment of this invention is the ability of LAC to induce
apoptosis mostly in activated and proliferating cells and not in normal
resting cells. This
has value in eliminating cancerous cells and antigen-specific T cells. The
elimination of
the latter will create a specific immune tolerance to alloantigens in
transplantation, and to
selfantigens in autoimmune diseases.
The Effect of LAC is Rapid and Reversible
CA 02372316 2002-02-21
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We next investigated how fast and how long LAC could exert its
effects on the lymphocytes, since this information is necessary to assess the
requirement of
the proteasome activity for events related
to cell activation and proliferation. PBMC were pretreated with LAC (10 pM) or
medium
for a period as indicated in Fig. 4A. The cells were then washed and
recultured in the
presence of PHA. The thymidine uptake was measured 3 days later. It was
clearly
demonstrated that 3h preincubation with LAC was sufficient to cause
significant inhibition
on the subsequent mitogen-stimulated proliferation in T cells, although 16h
preincubation
with LAC was more effective. This result indicates that LAC can enter the
cells rapidly
within 3h.
We used Jurkat cells that have high constitutive proteasome activity to
evaluate the duration of LAC's effect once the drug entered the cells. Jurkat
cells were
treated with LAC (6 ~zM) for 3h, which was sufficiently long for the compound
to enter
the cells as shown above. The cells were then thoroughly washed and
recuitured, and they
were harvested at Oh, Sh and 21h after the wash, and the proteasome activity
in the cell
lysates was measured using a chromogenic chymotrypsin substrate. We have
previously
established that the proteinase activity measured by this assay was
predominantly (more
than 90%) derived from the proteasome (Wang et al., 1997, Eur. J. Immunol.,
supra). As
shown in Fig. 4B, the proteasome activity in Jurkat cells was almost
completely inhibited
by 3h preincubation with LAC at 6 uM. Five hours after the LAC was washed out,
the
proteasome activity in the cells was still significantly inhibited but the
inhibition was
reduced compared with that at Oh. By 21 h, the proteasome activity returned to
a
near-normal level. It is to be noted that the short 3h treatment with LAC did
not affect the
viability of the Jurkat cells, and this is also reflected by the normal
proteasome activity of
the treated cells at 21h. The result shows that LAC is not stable and loses
its activity
within 21 h in the cells.
We also investigated whether LAC was stable in the culture
supernatant. LAC (6 pM) was added to Jurkat cells culture for 4h, 6h, 16h or
24h. The
CA 02372316 2002-02-21
-28-
conditioned medium was harvested and used to treat fresh Jurkat cells for 3h,
and then the
proteasome activity in the lysates of the fresh Jurkat cells was assayed. As
shown in
Fig. 4C, 4h to 24h conditioned media without LAC did not affect the proteasome
activity
of the fresh Jurkat cells. The media conditioned with LAC up to 6h could still
actively
inhibit the enzymatic activity, but after 16h, the LAC-conditioned media lost
their
inhibitory effect. The loss of LAC activity in the l 6h and 24h conditioned
medium is
unlikely due to trapping of LAC by proteasomes released by dead Jurkat cells,
because
LAC could rapidly enter the live cells and the equilibrium of the LAC
concentration
between both sides of the cytoplasmic membrane should be established very
fast. Thus,
the proteasomes whether released or not should not make a difference in terms
of trapping
LAC. Besides, we have also noticed that LAC kept in cell free culture medium
at 4°C
would lose its activity within 24h (data not shown). These results indicate
that LAC is not
only unstable within the cells, but is also unstable in the supernatant.
LAC's capability to enter the cells to inhibit the proteasome activity
rapidly (less than 3h), and its short active duration within the cell and in
the culture media
(about 16h) makes the compound a very useful reagent to evaluate the
requirement of the
proteasome activity in various events during cell activation and
proliferation, since we
could pinpoint the period when the proteasome activity is critical.
It is an embodiment of this invention, the use of LAC can be regulated
in a time course sequence to be most effective at the period when proteasome
activity is
critical to maximise the effect of LAC on cells.
Proteasome Activity is Required for IL-2Ra Upregulation
In the four systems of T and B cell activation and proliferation studied
in the first section, the growth promoting activity of IL-2 is indirectly (for
stimulation by
PHA, anti-CD3, and anti-CD28 plus ionomycin), or directly (for SAC plus IL-2)
involved.
We then investigated the role of proteasome in IL-2Ra expression and IL-2
production. As
shown in Fig. 5, CD25 was upregulated in CD3+ T cells 40h after stimulation
with PHA.
When LAC (10 uM) was added in the beginning of the culture, the upregulation
was
CA 02372316 2002-02-21
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significantly inhibited. On the other hand, IL-2 production by PBMC 2 to 4
days after
PHA stimulation in the absence or presence of LAC (10 uM, added at the
beginning of the
culture) was also examined, but no consistent difference was found (data not
shown).
Under the experimental condition used, the viability of the LAC-treated cell
was
reasonable (>80% at 40h) as described in the previous section as LAC was added
only
once initially. Moreover, no consistent change of IL-2 production in LAC-
treated cells was
a functional indication that the cell viability was reasonable and is not of a
concern in
interpreting the data. The results from this section indicate that IL-2Ra
upregulation but
not IL-2 , production is proteasome-dependent, and the suppressed IL-2Ra
expression
likely contributes to LAC's inhibitory effect on T cells activation and
proliferation.
The Proteasome Activity is Critically Required Between Go and GIIS Boundary in
T
Cells
Like normal T cells, the proliferation of Jurkat cells was also potently
inhibited by LAC (data not shown). We used synchronized Jurkat cells to
identify the
LAC-sensitive phases) of the cell cycle. Jurkat cells were first synchronized
at the G2/M
boundary by nocodazole (Fig. 6A). The cells were released from the blockage by
washing
out nocodazole. In the control sample, more than half the cells traversed
through the M
phase and arrived at the G: phase within 4h. In the test sample, LAC (6 uM)
was added to
the culture 3h before the release, so the compound could have enough time to
enter the
cells. LAC was also added to the culture after the release. However, the
Jurkat treated with
LAC traversed through the M phase to the Gl phase at a similar pace as the
control cells.
Since the total duration of the assay was around '71i (3h preincubation plus
4h after the
release), LAC was certainly active during this period. The fact that most of
synchronized
Jurkat cells could traverse through G2/M to Gl in the presence of LAC for 7h
again
suggests that the viability of the cells thus treated is not a matter of
concern. This result
shows that the G2 to Gl progression is not proteasome-dependent.
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We next studied requirement of the proteasome activity for the
progression from the Gl/S boundary to the G2/M phase. The Jurkat cells were
synchronized at the GINS boundary by HU blockage. The cells were then released
by
washing out HU. Within 9-12h, the majority of the cells progressed to the S
and G2/M
phase (Fig. 6B). When LAC was added to the culture immediately after the
release, it
slowed but did not block the cell cycle progression from the Gl/S boundary to
the G2/M
phase, as evidenced by the histograms at 6h and 9h post the release. It is to
be noted that
although the percentage of cells in the SlG2lM phase in the LAC-treated sample
was
similar to that of controls (the inset table of Figure 6B), the peak of
fluorescence was
lagged behind (histogram array). Beyond 9h, the cells gradually lost their
synchronization,
the viability of the cells started to decline and LAC gradually lost its
activity, so the data
became difficult to interpret. The result from this part suggests that the
proteasome activity
is required for optimal progression from the G11S boundary to the G2/M phase,
because the
progression could still proceed albeit at a slower pace when the proteasome
activity is
inhibited. The result also implies that the absolutely proteasome-dependent
window during
the cell cycle, as evidenced by the near-total inhibition of S phase entry in
LAC-treated
mitogen-stimulated lymphocytes according to the proliferation data, must be in
the Gl
phase before the target point of HU, which inhibits ribonucleotide reductase
in the G./S
boundary (Brown et al., 1996, Cell 86:517).
The cycling Jurkat cells are obviously not the best model to study the
events in the Gi phase since the G2/M synchronization become desynchronized by
the time
the cells re-enter the S phase, and there is no appropriate method to
synchronize the Jurkat
cells at the early Gi phase. We therefore decided to use mitogen-stimulated
normal T cells
to study the role of the proteasome in the G1 phase.
T cells from PBMC were at Go when isolated. After 16h stimulation
with PHA, they remained before the S phase (Fig. 6C). At 40h, about 20% of the
cells
were in the S and G2/M phases. The peak of 3H-thymidine uptake according to a
16h pulse
was between 48h and 64h (data not shown), although at 64h, the cells in the S
and G2/M
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phases were still about 20% (Fig. 6C). The lack of an increase in percentage
of cells in the
S and G2/M phases at 64h compared with that at 40h was likely due to the exit
of the cells
from the S and G2/M phase. It is to be noted that the cycling T cells in this
system never
reaches 100%, because about 15% of the cells were non T cells, and an
additional 20%
were non responsive T cells. Taken the cell proliferation and cell cycle
analysis together,
the Gl/S boundary of the cycling T cells should be between about 35h and 48h
after the
PHA stimulation. The boundary was broad because the synchronization was not
ideal.
In this model, the role of the proteasome in the S phase entry was
examined. As shown in Fig. 6C, LAC added once at 16h could totally block the S
phase
entry when examined at 40h. We have noticed that when the cell viability was
evaluated at
40h, there was an increase of cell death comparing the 16h addition of LAC
with the Oh
addition (about 25% vs about 17%, data not shown). The increased cell death
was also
reflected in the cells with < 2N DNA in the 40h histogram. However, such a
viability was
still reasonable and would not invalidate our conclusion. According to 3H-
thymidine
uptake, LAC was strongly inhibitory even added as late as 40h (Fig. 6D).
However, no
difference on the percentage of the population in the S and G2/M phase was
observed at
64h whether or not LAC was added at 40h according to flow cytometry (Fig. 6C).
The
discrepancy could~be explained by the fact that the 20% cells were already in
the S and
G2/M phases at 40h when LAC was added. LAC prevented additional cells from
entering
into the S phase, therefore the lack 3H-thyrnidine uptake. At the same time,
the drug
slowed the cell cycle progression from the Gl/S boundary to the G2/M phase,
hence the
lingering population in the S and G2/M phases according to flow cytometry.
It is worth mentioning the inhibition of proliferation by LAC was a
combinatory effect of cell cycle progress and cell death, the latter possible
being the
consequence of the former. The later the compound was added when more T cells
are
activated, a larger proportion of the effect should be attributed to cell
death caused by
LAC. The extensive cell death for the sample treated with LAC at 40h was not
fully
CA 02372316 2002-02-21
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reflected in the flow cytometry (Fig. 6C) as cells with less than 2N DNA. This
was due to
that the histogram was gated on a region of largely viable cells.
The results from this section indicate that the proteasome activity is not
required from the G2/M to the Gl phase. It optimizes the progression from the
GINS
boundary (as defined by the hydroxyurea target point) to the GZ/M phases, and
it is
absolutely required for the progression from the Go to the S phase.
In a specific embodiment of this invention LAC is used to reverse
ongoing graft rejection during a rejection episode. Most immunosuppressive
drugs do not
have the capability to reverse rejection once it begun. The use of LAC
overcomes the prior
art.
The Proteasome Activity is Essential for CDK2 but not for CDK4 Function
Cyclin-dependent kinases (CDK) are critical for cell proliferation.
CDK4 is essential in the early to mid-Gl phase to facilitate the S phase entry
(Tam et al.,
1994, Oncogene 9:2663; Lukas et al., 1995, Oncogene 10:2125) and CDK2 is
critical in
the late Gl as well as throughout the S phase for the cell cycle progression
(Van der
Heuvel et al., 1993, Science 262:2050). We therefore examined the role of the
proteasome
in CDK4 and CDK2 activities in mitogen-stimulated T cells. In all the models
used in this
section, LAC was added only once at the beginning of the culture.
Consequently, the
viability of the LAC-treated cells was good for the first 16h and was
reasonable at 40h,
and was not a factor that might interfere with the interpretation of the
results.
As shown in Fig. 7A, the resting T cells had some CDK4 activity, and
the activity reached a plateau within 16h of the activation. This was in
agreement with the
critical role of CDK4 in the early G phase. Inhibition of the proteasome
activity by LAC
from 0-16h (LAC added once at Oh) did not affect the CDK4 activity when
examined at
16h and 40h (Fig. 7A). This indicates that the induction and maintenance of
CDK4 activity
during the G1 phase is not proteasome-dependent.
In contrast to CDK4, the CDK2 activity was augmented at 16h but the
augmentation was more prominent at a later stage close to 40h after the
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mitogen-stimulation (Fig. 7B), and this reflected its essential role starting
from the late Gl
phase and extending to the early S phase. The presence of LAC from Oh to 16h
(LAC
added once at Oh) significantly inhibited CDK2 activity at 16h and more so at
40h.
Therefore, the protea.some activity during the early activation stage (Oh-16h)
is essential
for the activation of the kinase at the G1 phase and early S phase. The
unchanged CDK4
activity in the LAC-treated cells at 40h served as an internal control for the
repressed
CDK2 activity and indicating the latter was not due to the viability problem.
Since at the late Gl phase Cyclin E is the predominant partner of CDK2
(Sherr, 1993, Cell 73:1059), we next examined the Cyclin E-associated CDK
activity. As
shown in Fig. 7C, in spite that the Cyclin E protein was increased after the
LAC treatment
(LAC added once at Oh), the Cyclin E-associated kinase activity was almost
completely
inhibited by LAC. These results indicate that the CDK2 activity, and most
likely the
Cyclin E-associated CDK2 activity in the late G1 phase is proteasome-
dependent. The
results also suggest that the inhibition of the CDK2 activity is probably an
important
mechanism accountable for the LAC's effect in blocking the S phase entry.
It is an embodiment of this invention to have elucidated a downstream
target for proteasome activity. That is CDK2, more specifically Cyclin E-
associated CDK2
activity. It is also provided that with this knowledge, inhibitors of CDK2
can. be used alone
or in combination with proteasome inhibitors. It is further provided that the
aforementioned compositions are of a pharmaceutically effective amount to
induce
apoptosis or for any other cellular or physiological effect. Since CDK4
activity is
important in Go to G1, progression and it is not affected by proteasome
activity, it is
conceivable that inhibitors for CDK4 can be used in combination with
proteasome
inhibitors of a pharamceutically effective amount to achieve additive effect
in blocking
cell proliferation and in any other relevant cell function.
Inhibitors in this application are defined as any element capable of
silencing the activity of a protein at the level of gene transcription,
translation, or post-
translational modification of the protein as well as elements capable of
interfering with the
CA 02372316 2002-02-21
t
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protein. These may include but are not limited to antibody or other ligands,
anti-sense or
antagonist molecules.
Degradation of Cyclin E but not Cyclin A is Proteasome-Dependent
It is a specific embodiment of this invention that contacting LAC with
CDK2 is inhibitory to CDK2 activity, more particularity it is the inhibitory
effect of LAC
on Cyclin E. The inhibitory effect of LAC is the disruption of cell cycling.
Oscillation of cyclins during the cell cycle is a mode of regulation for
the CDK activities. Since the CDK2 activity is proteasome-dependent, and CDK2
associates predominantly with Cyclin E and cyclin A at the G1/S boundary and
during the
S phase respectively (Pagano et al., 1992, EMBO J. 11:961; Hall et al., 1995,
Oncogene
11:1581), we studied the role of the proteasome in degradation of these two
cyclins. As
shown in Fig. 8A, the Cyclin E level was apparently increased around 40h after
PHA
stimulation of the T cells, which were then at the G1/S boundary. If the
activated cells
were treated with HU, the Cyclin E level was significantly enhanced comparing
with those
treated with PHA alone (Fig. 8A). This reflects a better synchronization at
the Gl/S
boundary by HU, and was consistent with our knowledge that the Cyclin E level
peaked at
the boundary. After the boundary, the Cyclin E level started to decline, and
the decline
was prevented by LAC (Fig. 8A). This clearly demonstrates that the degradation
of
Cyclin E is a proteasome-dependent process, although whether the increased
Cyclin E
level contributes to LAC's effect on the cell cycle is a matter of debate.
For cyclin A, the level was increased around the late Gl phase after the
mitogen stimulation as shown in Fig. 8B. The blockage of the cycle at the Gl/S
boundary
with hydroxyurea did not further increase the cyclin A level. However, when
the cycle
passed the boundary and entered the S phase, the cyclin A level was
significantly
augmented (Fig. 8B), consistent with the notion that cyclin A is mainly an S
phase cyclin.
Unlike that of Cyclin E, the level of cyclin A did not decline during the S
phase and LAC
did not affect the level during this period. This suggests that the proteasome
is not
involved in cyclin A degradation, at least in the Gl and S phases, and that
LAC's effect on
CA 02372316 2002-02-21
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inhibiting cell proliferation is unlikely mediated via the cyclin A levels.
The Gl/S phase
synchronized T cells represented activated cells, and prolonged exposure to
LAC would
cause significant cell death. However, 6h treatment of LAC did not apparently
affect the
cell viability, while the blockage of Cyclin E degradation but not cyclin A
degradation was
obvious at that time point. Moreover, cyclin A could be considered as an
internal control
for Cyclin E indicating that the LAC-induced cell death should not affect the
conclusion in
this section.
The Role of Proteasome in Regulating Levels of CDK Inhibitors p27K'pl and
p21~'pi
In a specific embodiment, LAC is capable of suppressing the up
regulaion of the CDK inhibitor p21~"pl and in blocking the degradation of the
CDK
inhibitor p27K'pl.
In addition to the cyclin levels, the GDK activities are also controlled
by several low molecular weight inhibitors. We have examined in this study the
effect of
the proteasome on the CDK inhibitors p27K'pl (Hall et al., 1995, supra) and
p2lo'pl (e1-
Deiry et al., 1993, Cell 75:817). As shown in Fig. 9A, the resting T cells had
a high level
of p27K'pl and the level decreased gradually when the cells moved to the Gl/S
boundary
40h after the mitogen-stimulation. This is in agreement with previous reports
(Hengst et
al., 1996, Science 271:1861; Nourse et al., 1994, Nature 372:570). The
presence of LAC
(added once at Oh) significantly blocked the decrease when assayed at 16h,
showing that
the degradation is a proteasome-dependent process. The blockage was less
obvious when
assayed at 40h, probably because the gradual loss of LAC activity during the
40h culture.
The result suggests that the blocking of p27K'pl degradation is a contributing
mechanism
contributing for the inhibitory effect of LAC on the CDK2 activity. Unlike
p27K1p1, p2lc'pl
had a low level of expression in resting T cells. The level was rapidly
augmented after 16h
PHA activation, and the high level was maintained at the Gl/S boundary at 40h
(Fig. 9B).
Such an induction suggests that p2lC'pl might be required in the G phase for
roles other
than a CDK inhibitor. Interestingly, LAC strongly suppressed the upregulation
of p21~'pi
in the Gl phase, indicating that the expression of p2lC'pl is proteasome-
dependent, and
CA 02372316 2002-02-21
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suggesting that the proteasome might facilitate cell proliferation via its
role in p2lC'pi
upregulation during the Gl .phase. In this experiment, LAC was only added once
at the
beginning of the culture, and the viability of the treated cells at 16h was
good (83~) and
should not be a concern in drawing the conclusion.
Disruption of Cell-Cell Interaction
Cell-cell interaction is essential in antigen presentation and in T cell's
help to T and B cells. The adhesion molecules are necessary to establish the
cell-cell
interaction. Blocking the adhesion molecules ICAM-1 and LFA-1 is known to
inhibit
immune responses and to suppress graft rejection. Our data clearly shows that
inhibition of
the proteasome activity will effectively interfere with the cell-cell
interaction during
lymphocyte activation in both human (Fig. 10) and mouse (Fig. 11) systems, and
the
upregulation of an adhesion molecule ICAM-1 is repressed by the proteasome
inhibitor
lactacystin (Fig. 12). Therefore, inhibition of the proteasome activity will
be a useful way
to control undesirable immune responses during graft rejection, autoimmune
diseases, and
inflammation.
Proteasome Activity is Required for Nitric Oxide Production
Nitric oxide (NO) produced by macrophages is involved in
inflammation and septic shock. We have shown that inhibition of the proteasome
activity
could effectively repress the endotoxin LPS-induced NO production (Fig. 13).
The
usefulness of proteasome inhibitors in inflammation and in septic shock is
implicated. Fig.
14 demonstrates that proteasome activity is required for NO synthase
expression. The
addition of LAC decreases the expression of mRNA for NO synthase.
The Effect of Proteasome on Mitochondria) Function
Mitochondria are pivotal organelles in the cells and their primary
function is to produce ATP via the Krebs cycle coupled to the oxidative
phosphorylation
of the respiratory chain. An intact function of mitochondria is also required
for proper cell
viability. Damage of the mitochondria) membrane potential or release of
cytochrome C or
CA 02372316 2002-02-21
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other apoptogenic factors from the mitochondria to the cytosol will induce
cell death via
apoptosis.
In our study, we have found that the electron transport in mitochondria
of Jurkat T lymphocytes is dependent on the intact activity of the proteasome.
A
proteasome-specific inhibitor lactacystin (LAC) could rapidly (within 4h)
reduce the
electron transport at the complex IV of the respiratory chain, and the effect
could be
reversed by adding back exogenous cytochrome C (cytoC).
In Fig. 15, the respiration of Jurkat cells treated with LAC for 4h (curve
4) but not for 2h (curve 3) could not be resumed by adding succinate after
Complex I
blockage, and CCCP failed further to stimulate the respiration as it could in
control Jurkat
cells and in rat mitochondrial preparation (curves 5 to 6, respectively).
Adding. rat kidney
mitochondria to the blocked reaction results in normal respiration (curve 4),
showing the
reagents and the oxygen electrode are functional. The results indicate that
LAC
compromises the electron transport after Complex I.
In Fig. 16, Jurkat cells treated with LAC for 2h (curve 3) had similar Oz
consumption after Complex III, like that of untreated Jurkat cells (curve 2)
and rat kidney
mitochondria (curve 1). After 4h LAC treatment, the 02 consumption of the
Jurkat cells
could not be resumed by ascorbate and TMPD to a level similarly high as that
of untreated
Jurkat and rat mitochondria, and the decoupling reagent CCCP had no effect in
the treated
cells (curve 4). Adding back rat kidney mitochondria into the assay could
resume the 02
consumption, showing a functional assay system. Curves 5 to 6 are untreated
Jurkat cells
and rat kidney mitochondria, respectively, showing normal function of Complex
IV. This
result shows that the LAC treatment caused compromised function in the
electron
transport at Complex IV.
In Fig. 17, Jurkat cells treated with LAC (curve 3) have reduced
augmentation of 02 consumption after the addition of ascorbate and TMPD,
compared
with untreated Jurkat cells (curve 2) and rat kidney mitochondria (curve 1).
FCCP could
not further stimulate the respiration, as it could in normal Jurkat cells and
rat kidney
CA 02372316 2002-02-21
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mitochondria. When exogenous cytochrome c (CytoC) was added to the LAC-treated
cells, the respiration resumed to a rate similar to that of untreated Jurkat
cells and
mitochondria. CytoC had no additive effect in stimulating respiration in
normal Jurkat
cells and rat mitochondria (curves 2 and 3, respectively).
The implication of aforementioned findings is as follows:
In hyperthyroidism, the mitochondria) activiy is overactive due to the
effect of the thyroid hormone. This results in many symptoms such as excessive
body heat,
and imbalance of energy uptake and consumption. The proteasome inhibitors
could reduce
the rate of mitochondria) respiration and have therapeutic effect to this
disease.
In fast-growing cells such as cancer cells or activated lymphocytes, the
mitochndria are more active than in normal cells in order to meet the energy
requirement
of a high metabolic activity of these cells. Consequently, inhibition of the
mitochondria)
respiration could curb the proliferation of the cancer cells or activated
lymphocytes while
have less detrimental effects to normal resting cells. In addition, apoptosis
could be
induced in the cycling cells but not resting cells. Thus, inhibition of the
proteasome
activity will have therapeutic effect in cancer and in diseases involving
lymphocyte
activation and proliferation, such as seen in graft rejection and autoimmune
diseases.
Rapid Assays for A High Through-Put Screening Procedure to Identify Additional
Proteasome Inhibitors
In our study, we have shown that about 70-80% of the chymotrypsin-
like activity in the lymphocyte lysates is derived from the proteasome (Fig.
20). In a
positive control, LAC at 10 uM could inhibit 90% of the 20S proteasome
activity which
was in a range similar to that of the cell lystates. Increasing the
concentration of LAC to
20 uM did not further increase the inhibitory effect, suggesting that the LAC
concentration
used was already saturating. The remaining 10% activity might be derived from
non-
proteasome proteinases in the 20S proteasome preparation. When 10 uM LAC was
added
to the 70-h cell lysate, it inhibited 73.4%, 76.7% and 86.7% of total
chymotrypsin-like
activity in the lysates from medium-, PHA- and PHA plus R.APA-treated PBMC,
CA 02372316 2002-02-21
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respectively, and those percentages represented the portion of enzymatic
activity from the
proteasome.
The implication of this finding is that mammalian cell lysates without
other purification could be used as a convenient source of proteasomes. Tagged
substrates
specific for the known proteasome activities, such trypsin-like, chymotrypsin-
like, and
PGPN activities can be used as displaying parameters. Known compounds could be
added
into this enzyme/substrate system, and the compounds) that inhibits) one or
several
aforementioned enzyme activities of the lysate above a certain threshold (for
example
40%) will be identified as proteasome inhibitors. These assays could be
modified to use
purified or partially purified 20S or 26S proteasome as a source of the
proteasome
enzymes. Since such assays are simple (only three components) and rapid (only
several
minutes of reaction period), they could be adapted for high through-put
screenings, and
included in a kit format.
The Effect of Immunosupressive Drugs on Proteasome Function
Rapamycin (R.APA) is a potent immunosuppressive drug, and certain of
its direct or indirect targets might be of vital importance to the regulation
of an immune
response. Seven RADA-sensitive genes are known and one of them encoded a
protein with
high homology to the a subunit of a proteasome activator (PA28a). This gene
was later
found to code for the ~i subunit of the proteasome activator (PA28 Vii).
Activated T and B
cells had upregulated PA28 ~i expression at the mRNA level. Such upregulation
could be
suppressed by RAPA, FK506, and cyclosporin A (CsA). RAPA and FK506 also
repressed
the upregulated PA28a messages in PHA-stimulated T cells. At the protein
level, RAPA
inhibited PA28a and PA28 ~i in the activated T cells according to
immunoblotting and
confocal microscopy. Probably as a consequence, there was a fourfold increase
of
proteasome activities in the PBMC lysate after the PHA activation. RAPA could
inhibit
the enhanced part of the proteasome activity. Considering the critical role
played by the
proteasome in degrading regulatory proteins, a proteasome activator is a
relevant and
CA 02372316 2002-02-21
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important downstream target of rapamycin, and that the immune response could
be
modulated through the activity of the proteasome.
A lot of efforts have been made to identify direct targets of RAPA. It is
now known that RAPA complexes with a 12KD FK506-binding protein (FKBP 12)
(Harding et al., 19$9, Nature 341:371; Siekierka et al., 1989, Nature
341:755). The
RAPA-FKBP12 complex then binds to cytoplasmic proteins termed TORT and TOR2
(target of rapamycin) in yeast (Kunz et al., 1993, Cell 73:585; Helliwell et
al., 1994, Mol.
Biol. Cell. 5:145), and FRAP and RAFT1 in mammalian cells (Brown et al., 1994,
Nature
369:756). These target proteins have high degree of homology in their primary
sequences,
and their C-terminal sequences share certain homology with catalytic domains
of both PI-3
kinase and PI-4 kinase.
The mRNA expression of most genes so far studied, whether they are
constitutively expressed or induced after stimulation, are not sensitive to
RAPA (Tocci et
al., 1989, J. Immunol. 143:718; Shan et al., 1994, Int. Immunol. 6:739). It
follows that the
genes that are sensitive to RADA at the mRNA level have a good probability of
being
secondary taxgets of RADA and being pivotal in controlling the immune
response.
Expression of PA28 ~3 at mRNA and protein levels was found to be sensitive to
RADA, so
was that of the PA28a subunit which shares a high degree homology with PA28~i.
It was
found that proteasome activity was repressed by the drug.
In HeLa cells, PA28 ~i expression was dramatically upregulated at the
mRNA level by IFNy treatment after 24h. This was similar to the regulation of
PA28a
(Realini et al., 1994, supra). When human tonsillar T cells were stimulated by
PHA, the
PA28 ~i expression was augmented after 20h, and the augmentation could be
suppressed by
IOnM RADA as expected (Fig. 18A). In addition, the expression was also
sensitive to CsA
(1 yaM) and FK506 (lOnM). In tonsillar B cells, SAC and IL-2 upregulated the
PA28~i
mRNA expression, and RAPA was inhibitory (Fig. 18B). Similarly, the mRNA
expression
of PA28a, which has a high degree of homology with PA28 ~3, was upregulated in
CA 02372316 2002-02-21
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PHA-activated T cells, and the upregulation was repressed by FK506 and RAPA
(Fig.
18C).
Expression of PA28 ~ and PA28a at the protein level was also
examined. The result of immunoblotting demonstrated that the activated T cells
had
increased PA28 ~i compared with resting T cells, and the increase was
inhibited in the
presence of RAPA (Fig. 19A). Since the anti-PA28a antiserum did not seem to
recognize
the denatured proteins, we used confocal immunofluorescent microscopy to
examine the
PA28a protein as well as the PA28 ~i protein in the T cells. The experiment
was carried out
in an one-way blind fashion, the microscopy operator without being informed of
the
treatment of the cells. As shown in Fig. 19B, RAPA plus PHA-treated T cells
had
significantly lower levels of both PA28a and PA28(3 proteins compared with T
cells
treated with PHA alone. We have noticed that although the difference between
the
PHA-activated T cells in the absence and presence of RAPA was highly
significant
(p<0.0001 ), the difference of the numeric values of the mean fluorescence
intensity
between the two types of cells, especially in the case of PA28 (3, was rather
small.
However, there was a high standard deviation in the PHA-treated samples. A
closer
inspection revealed that about 40% of the cells treated with PHA alone had
elevated
PA28 (3 and PA28a signals while the rest had basal level expression. This
caused the high
standard deviation. Considering that there were 20% non T cells in the T cell
preparation,
and that PHA does not activate all the T cells in the culture simultaneously,
those 40%
cells with the high signals probably represented the truly activated T cells.
Therefore, the
actual difference between the activated. and drug-repressed cells could be
much bigger
than the data presented in the histogram.
Taken together, our data indicates that RADA inhibits the expression of
PA28a and PA28(3 at both mRNA and protein levels. The inhibition of the PA28
mRNAs
is a likely cause for the observed decrease of the corresponding proteins.
However, we
could not exclude the possibility that RAPA might also act directly at the
translation level
for PA28a and PA28~i.
CA 02372316 2002-02-21
-42-
In as much as PHA could upregulate and RADA could repress
expression of the proteasome activator PA28 ~3 and PA28a in the T cells, it is
logical to
examine changes of proteasome activity in these cells. PBMC lysates were
assayed for
their proteinase activity at pH 8.2 which favors the proteasome activity,
using a
chymotrypsin substrate as a representative parameter. Forty and seventy hours
after
stimulation by a T cell mitogen PHA, the chymotrypsin-like activity in the
PBMC
increased 2.1 fold and 3.8 fold, respectively (Fig. 20A). RADA at l OnM
repressed 23.1
and 41.1 % the activity in the PBMC, respectively, at these time points.
We then tried to determine the part of enzyme activity in the lysates
conferred by the proteasome. In a positive control, LAC at 10 uM could inhibit
90% of the
20S proteasome activity which was in the range similar to that of the cell
lysates (Fig.
20B). Increasing the concentration of LAC to 20 uM did not further increase
the inhibitory
effect, suggesting that the LAC concentration used was already saturating. The
remaining
10% activity might be derived from non-proteasome proteinases in the 20S
proteasome
1 S preparation. When 10 uM LAC was added to the 70h cell lysate, it inhibited
73.4%, 76.7%
and 86.7% of total chymotrypsin-like activity in the lysates from medium-, PHA-
and
PHA plus RAPA-treated PBMC, respectively, and those percentages represented
the
portion of enzymatic activity from the proteasome (Fig. 20B). The net
proteasome activity
increased by 4 fold from 42.6 x 103 units/20 ug protein in unstimulated cells
to 170.3 x 103
units/20 ~g protein in the PHA-activated cells. In RAPA-treated cells, the
activity
decreased to 113.2 x 103 units/20 pg protein. This equated to 33.6% inhibition
of the total
activity, or 44.7% of the augmented proteasome activity in the PHA-treated
PBMC. It is
therefore demonstrated that RAPA could inhibit the enhanced proteasome
activity during
T cell activation.
It is an embodiment of this invention to have identified known
immunosupressive drugs including rapamycin, FK506 and cyclosporin A as
inhibitors of
enhanced proteasome activity. It is therefore a specific embodiment of this
invention for
providing these immunosupressive drugs of a pharmaceutically effective amount
and in
CA 02372316 2002-02-21
- 43 -
combination with specific proteasome inhibitors of a pharmaceutically
effective amount,
as an example but not limited to LAC or its analogues to achieve an additive
effect in
blocking cell proliferation and any other relevant cell function. Such
combinations as
described can be used but are not limited to the treatment of cancer, graft
rejection and
S autoimmune diseases.
Elimination of alloantigen-specific response
The results of the functional assay shown in Figure 21 suggests, that
there is clonal deletion of BALB/c-specific T cells when proteasome activity
of
alloantigen-activated T cells are inhibited for a brief period. The
consequences of this
finding suggests that proteasome inhibitors can be administered when specific
T cells are
activated, thereby potentially eliminating the activity of specifically
activated T cells while
leaving non-activated T cells intact: It is therefore an embodiment of this
invention to use
proteasome inhibitors, particularly lactacystin in transplantation and
autoimmune diseases
where certain undesirable activated T cells can be repressed or eliminated and
the rest of
the T cell population is generally unaffected by such inhibitors.
The effect of caspase inhibitor zVAD.fmk. on LAC-induced DNA fragmentation
The effect of lactacystin as an apoptotic agent in Jurkat cells is shown
in Figure 22, by the typical apoptotic sign of DNA laddering. Addition of the
broad
spectrum caspase inhibitor zVAD.fins demonstrated an inhibitory effect on DNA
fragmentation that is concentration responsive. This result indicates that the
lactacystin-
induced apoptosis in Jurkat cells is caspase-dependent.
The effect of lactacystin on a pro-apoptotic Bcl-2 family member, Bik
The results shown in Figure 23 panel A, show that Bik, Bax, Bak, and
Bad are predominantly located in the mitochondria) fraction. Treatment with
lactacystin
does not appear to have altered the amounts of Bax, Bak and Bad (Fig. 23
panels A and
B). There is however a demonstable increase in the amount of Bik in the
lactacystin
treated Jurkat cells after 4 h, 5 h and 7 h (the first row of panels A and B),
when compared
CA 02372316 2002-02-21
-44-
with untreated cells. The results shown in Fig. 23, suggests that under normal
circumstances, Bik is degraded rapidly by the proteasome. Blocking of this
degradation
by a proteasome inhibitor; allows the pro-apoptotic Bcl-2 member to
accumulate. The
accumulation of Bik may possibly tip the balance between pro- and anti-
apoptotic factors
favoring apoptosis.
The effect of overexpression of Bcl-xL, an anti-apoptotic Bcl-2 family member
The human B cell line Namalwa stably transfected with an anti-
apoptotic Bcl-2 family member Bcl-xL, was shown to be more resistant to the
proteasome
inhibitor lactacystin than the untransfected, wild type Namalwa cells. The
results shown in
Figure 24 indicate that the transfected cells have demonstrably less DNA
fragmentation at
the different intervals and lactacystin concentrations tested. This suggests
that the
overexpression of Bcl-xL protein has probably counteracted the effect of the
accumulation
of the pro-apoptotic Bik. In this manner the Namalwa cells are somewhat
protected from
undergoing apoptosis.
In an additional experiment, Jurkat cells, wild type Namalwa cells and
Bcl-xL transfected Namalwa cells were treated with staurosporine and
lactacystin for 6 H.
Proteins from the mitochondria) fraction of these cells were analyzed by
immunoblotting
for the amount of Bik; Bcl-xL, Bax, and Bak. The results summarized in Figure
25, show
that Bik accumulates in the Namalwa cells (panel B, lane 3) and Jurkat cells
(panel A lane
2) after a 6 hour lactacystin treatment. 'This accumulation is due to the
inhibition of
proteasome activity and indicates that the degradation of Bik via the
proteasome is a
general phenomenon. The elevated amount of Bik, is likely a mechanism of
lactacystin-
induced apoptosis in the Jurkat and Namalwa cells. The accumulation of Bik was
only
observed in the lactacystin-treated but not in staurosporine treated cells,
eventhough
staurosporine could equally induce apoptosis in these cells. The expression of
exogenous
anti-apoptotic Bcl-2 member Bcl-xL as expected, was not detected in Jurkat
cells and wild
type Namalwa cells (panels A and B). The Bcl-xL overexpression was obvious in
the
CA 02372316 2002-02-21
- 45 -
transfected Namalwa cells (panel C). Moreover, there was an accumulation of
Bcl-xL
after lactacystin treatment, showing that under normal circumstances the
degradation of
Bcl-xL, like Bic is also rapid and depends on proteasome activity. These
results suggest
that the Bcl-xL-transfected Namalwa cells have two mechanisms to protect them
from
proteasome inhibitor-induced apoptosis. First the overexpression of the anti-
apoptotic
Bcl-xL changes the balance between pro- and anti-apoptotic factors and favors
the anti-
apoptotic factors. Second, after treatment with lactacystin, there is an
accumulation of Bcl-
xL which imparts additional weight to the anti-apoptotic factors.
Thus, the balance between the pro- and anti-apoptotic factors in cells is
crucial in deciding, the fate of these cells. Certain apoptosis-related
factors have a short
half life and their degradation is via the proteasome machinery. Therefore,
modulating the
proteasome activity with proteasome inhibitors is a useful way to control the
balance
between the pro- and anti-apoptotic factors. This control provides the means
to induce
cells into apoptosis or continued survival.
Accordingly, it is an additional embodiment of this invention to provide
the means to balance between pro-apoptotic and anti-apoptotic factors in a
cell using
proteasome inhibitors, particularly lactacystin.
DPBA is effective in treating ongoing heart allograft rejection in mice
The proteasome inhibitor DPBA could effectively reverse the ongoing
rejection. With a short-term treatment between day 3 and 6, the graft survival
was
prolonged to more than 13 days and is still counting.
The present invention is illustrated in further detail by the following
non-limiting examples.
CA 02372316 2002-02-21
-46-
EXAMPLE 1
Reagents
RPMI 1640, FCS, penicillin-streptomycin, and L-glutamine were purchased from
Life
Technologies (Burlington, Ontario, Canada). Lymphoprep was purchased from
NYCOMED (Oslo, Norway). PHA, hydroxyurea, nocodazole, and histone Hl were from
Sigma (St. Louis, MO). Staphylococcus aureus Cowan I (SAC) were obtained from
Calbiochem (La Jolla, CA), and lactacystin from Dr. E.J. Corey (25). Human rIL-
2 was
from La Roche (Nutley, NJ), and anti-CD3 mAb OKT3 was from ATCC (Rockville,
MD).
FITC-conjugated anti-CD3 mAb(clone SFCIRW2-8C8) and PE-conjugated anti-CD25
mAb (clone IHT44H3) were from Coulter (Miami, FL). Anti-CD28 mAb (clone 9.3)
was a
gift from Dr. P. Linsley (26). A fluorogenic chymotrypsin substrate SLLVY-MCA
was
from Peninsula Laboratories (Belmont, CA). Rabbit antisera against cyclin A,
Cyclin E,
p27K'pi, p2lc'pi, CDK2 and CDK4 were purchased from Santa Cruz Biotech (Santa
Cruz,
CA). [Y-Sap]ATP (3000 pCi/mmol) and [I2sI] protein A (30mCi/mg protein) were
ordered
from Amersham (Oakville, Ontario, Canada), and [Methyl-3HJ thymidine
(2Ci/mmol) was
from ICN (Irvine, CA).
~P~~ l'.11~'F11Y?
Peripheral blood mononuclear cells {PBMC) and tonsillar T cells were prepared
as
described before (Luo et al., 1992, Transplantation 53:1071; Luo et al., 1993,
Clin. & Exp.
Immunol. 94:371). The cells were cultured in RPMI 1640 supplemented with 10%
FCS,
L-glutamine and antibiotics. 3H-thymidine uptake was carried out as described
previously
(Luo et al., 1992, supra; Luo et al., 1993, supra).
DNA fragmentation assay
The assay was performed according to a protocol described by Liu et al (Liu et
al., 1997,
Cell. 89:175) with some modifications. Briefly, 2-6 million cells were re-
suspended in
50 u1 PBS followed by 300 u1 lysis buffer (100 mM Tris-HCI, pH 8.0, 5 mM EDTA,
0.2
CA 02372316 2002-02-21
-47-
M NaCI. 0.2% w/v SDS, and 0.2 mg/ml proteinase K). After overnight incubation
at 37°C,
350 u1 of 3M NaCI was added to the mixture and cell debris was removed by
centrifugation at 13000 g for 20 min at room temperature. DNA in the
supernatant was
precipitated with an equal volume of 100% ethanol. The pellet was washed with
cold 70%
ethanol and then dissolved in 20 ~1 of TE containing 0.2 mglml RNase A. After
incubation
at 37°C for 2 h, the DNA was resolved on 2% agarose gel and visualized
with ethidium
bromide staining.
Electron microscopy
T cells and Jurkat cells were examined by electron microscopy as described by
Tsao and
Duguid (Tsao et al., 1987, Exp. Cell Res. 168:365).
Flow cytometry for IL-2Ra
Two-color staining with FITC-anti-CD3 and PE-anti-CD25 was performed on
tonsillar
T cells. The method was described before (Luo et al., 1993, supra).
Proteinase assay
Jurkat cells were cultured with various treatments and were harvested and
sonicated in
300 ~1 PBS on ice for 40 sec. Twenty micrograms of protein per sample from the
cleared
lysates were supplemented to 100 u1 with 0.1 M Tris buffer (pH 8.2). The
fluorogenic
chymotrypsin substrate sLLVY-MCA was added at a final concentration of lOnM.
The
samples were incubated at 37°C for 15 min and the reaction was
terminated by adding 4 p1
2.5M HCI. The samples were then diluted to 2m1 with O.1M Tris pH 8.2, and
measured for
their fluorescence intensity by a PTI fluorometer (Photo Technology
International, South
Brunswick, NJ). The excitation wavelength was 384nm, and the emission
wavelength
440nm.
Cell cycle synchronization of T cells and Jurkat cells
Tonsillar T cells were cultured in the presence of 2 ug/ml PHA and 1mM
hydroxyurea for
40h. The cells thus treated were synchronized at the Gl/S phase. The
synchronization was
CA 02372316 2002-02-21
- 48 -
released by washing out hydroxyurea, and the cells were cultured in medium for
additional
6-22h according to the need of each experiment. The synchronization of Jurkat
cells was
described in our previous publication (Shan et al., 1994, Int. Immunol.
6:739). Briefly, the
Jurkat cells were starved in isoleucine deficient medium for 24h followed by
16h treatment
with 2mM hydroxyurea (HL~. Cells thus treated were synchronized at the G1/S
boundary.
For synchronization at the G2/M boundary, the Gl /S synchronized cells were
released
from hydroxyurea and cultured in regular medium for 6h, and then treated with
0.1 ~zg/ml
nocodazole for 16h. The cells were then synchronized at the G2/M boundary.
Cell cycle analysis
Flow cytometry was employed for cell cycle analysis for T cells and Jurkat
cells as
described before (Shan et al., 1994, supra) using propidium iodide staining.
Immunoblotting
Immunoblotting was employed to evaluate the levels of Cyclin E, cyclin A,
p2lC'pl and
p27K'pl. The general protocol was described in our previous publication (Chen
et al., 1996,
supra). Briefly, lymphocytes were lysed in the presence of proteinase
inhibitors. The
cleared lysates were quantitated for protein concentrations. An equal amount
of lysate
proteins (40 ug) of each sample was resolved by 10% SDS-PAGE and was
transferred to
PVDF membranes (Millipore, Bedford, MA). The membranes were then blocked with
5%
milk, and hybridized with rabbit antisera against Cyclin E, cyclin A, p27K'pl
and p2lC'pl at
dilutions suggested by the manufacturer. The signals on the membrane were
detected by
~lzsl~-protein A followed by autoradiography.
Immunoprecipitation and the kinase assay
Lymphocytes were lysed by a lysis buffer as used in the immunoblotting (Chen
et al.,
1996, supra), and cleared lysates were quantitated for their protein content.
For
immunoprecipitation, SO ~zl of rabbit antisera against CDK2, CDK4 or Cyclin E
were
added to the lysates equivalent to 20 or 40 ug protein depending on the
experiment. After
CA 02372316 2002-02-21
d
-
2h incubation at 4°C, the immune complexes were recovered by protein A-
conjugated
Sepharose (Pharmacia Biotech, Montreal, Quebec, Canada). The immune complexes
bound to protein A-Sepharose were extensively washed in a lysis buffer without
detergents
or EDTA, and resuspended in 50 u1 of kinase reaction buffer (100mM NaCI, 20mM
HEPES, pH7.S, SmM MnCl2, SmM MgCl2, 25 uM cold ATP, 2.5 uCi (y-32p] ATP, and
3 ug histone H1 as a substrate). The reaction was caxried out for 10 min at
room
temperature, and stopped by adding the SDS-PAGE loading buffer. After boiling
for 3
min, the samples were subjected to 10% SDS-PAGE. The proteins were then
transferred to
PVDF membranes and the signals were detected by autoradiography.
EXAMPLE 2
Assays Measuring Nitric Oxide Production
Macrophage Preparation and Culture
BALB/c mice were injected i.p. with 3m1 of 3% thioglycollate broth. Three days
later,
peritoneal exudate macrophages of the mice were harvested and washed at 170 g
for 10
min at 4° C. The macrophages were cultured in Teflon vials (2cm in
diameter) at
4x106/2ml with various reagents (LPS, 2 ug/ml; IFNy, 100u/ml; LAC, 0.62-5 ~M
for the
nitric oxide assay and 5 pM for the Northern blot assay).
Nitric Oxide Measurement
The nitrite concentration in the culture supernatant was measured as a way to
indirectly
reflect the nitric oxide level following a method described by Ding et al
(Ding et al., 1988,
J. Immunology 141:2407). Release of reactive nitrogen intermediates and
reactive oxygen
intermediates form mouse peritoneal macrophages: comparison of activation
cytokines and
evidence for independent production. Briefly, 100 u1 of supernatants collected
from 48h
macrophage cultures was incubated with an equal volume of the Griess reagent
(1%
sulfanimide/ 0.1 % naphthylethylene diamine dihydrochloride/ 2.5% H3P0~ )at
room
CA 02372316 2002-02-21
-S~-
temperature for 10 min in 96-well microtitration plates, the O.D. was measured
at SSOnm.
Sodium nitrite of various concentrations were used to construct standard
curves.
Northern Blot Analysis of iNOS Expression
The expression of inducible nitric oxide synthase at the mRNA level was
analyzed by
Northern blot as described in our previous publication (Shan et al., 1994,
supra). After an
overnight culture, the mouse macrophages were harvested and their total
cellular RNA was
extracted with the guanidine/CsCI method. The RNA (10 ug/lane) was resolved in
1%
agarose-formaldehyde gels and blotted onto nylon membranes. A 562-by fragment
corresponding to the mouse iNOS cDNA (Xie et al., 1992, Science 256:225) was
obtained
by reverse transcription/PCR using the mouse macrophages total RNA as
templates. The
fragment was labeled with 32P with random primers and used as a probe for the
Northern
blot.
FY A MP1.F 't
Respiration of Jurkat Cells
Preparation of mitochondria
Rat liver of rat kidney proximal tubules mitochondria were isolated by
differential
centrifugation in a medium containing 250 mM sucrose, 1 mM HEPES-Tris, 250 ~M
EDTA (pH 7.5). The last washing of the mitochondria was performed in the same
medium
without EDTA. Protein concentration of the mitochondrial suspension was
measured after
solubilization of the membranes in 0.1% SDS with the Pierce-BCA
(bicinchroninic acid)
protein assay reagent (Pierce, Rockford, IL, USA), using bovine serum albumin
as a
standard.
Respiration Measurements
The Jurkat Cells (JC) (30x106/ml) or rat kidney proximal tubules mitochondria
(RKM)
(0.5 mg of protein/ml) were incubated in 1 ml measuring chamber at 37°-
C in a respiration
buffer containing 200 mM sucrose, S mM MgCl2, 5 mM KH2P04, and 30 mM
CA 02372316 2002-02-21
-51-
HEPES-Tris (pH 7.5). During respiration experiments following substrates and
inhibitors
were used: 0.005% Digitonine (Dig); 10 mM Succinate (Suc); 1 mM Ascorbate
(Asc); 0.4
mM tetramethyl-p- phenylenediamine (TMPD); 1 uM CCCP, 1 uM FCCP; 0.1 uM
Rotenone (Rot); 50 nM Antimycin A (Anti); 1 mM KCN; 100 ~aM Cytochrome C (Cyt
C).
The respiration rate of the Jurkat Cells and mitochondria was measured
polarographically
with a Clarke oxygen electrode (Yellow Springs Instruments, Yellow Springs,
OH, USA)
using 1 ml thermojacketed chamber. Oxygen concentration was calibrated with
air-saturated buffer using Hypoxanthine - Xanthine Oxidase - Catalase system
("chemical
zero"). Oxygen consumption was continuously recorded using a "MacLab/8"
(Analog
Digital Instruments, USA) connected to a Macintosh SE computer and the MacLab
Chart
v.3.3.4 software. Rates of oxygen consumption are expressed as ng-atoms of
oxygen/min.
FYAMP1.F. d
The effect of immunosuppressive drugs
Cell culture
PBMC were prepared by Lymphoprep gradient as described before (Luo et al.,
1993,
supra; Shan et al., 1994, supra). Tonsillar T cells were prepared by one cycle
of SRBC
rosetting and such preparation contained 80-85% CD3+ cells. The remaining
tonsillar cells
were referred to as the tonsillar B cells, which were about 90% CD20+ cells.
Northern blot analysis
The method is described in our previous publication (Shah et al., 1994,
supra). Tissue or
lymphocyte total RNA was extracted with the guanidine/CsCI method and used in
the
Northern blot analysis. A 358-by fragment corresponding to positions -14 to
314 of the
PA28~i cDNA (Ahn et al., 1995, FEBS Lett. 366:37) from clone SF2 was labeled
with Sap
using random primers and was used as a probe for PA28 ~i messages. A 400-by
fragment
corresponding to positions between 267 and 666 of the PA28a cDNA (Realini et
al., 1994,
supra) was obtained with RT-PCR and was used as a probe for PA28a messages.
The 5'
CA 02372316 2002-04-15
-52-
and 3' primers for the RT-PCR were GAAGAAGGGGGAGGATGA (SEQ. ID. No. 1 )
and AGCATTGCGGATCTCCAT (SEQ ID No. 2), respectively.
Immunoblotting
T cell lysates (40 ug protein/sample) were separated on 12% SDS-PAGE, and
blotted onto
PVDF membranes. The membranes were then hybridized with rabbit anti-PA28(3
antiserum (Ahn et al. 1996, J. Biol. Chem. 271:18237) followed by'25I-protein
A. Detailed
methods were described previously (Chen et al., 1996, supra).
Confocal immunoflurescent microscopy
Cultured tonsillar T cells were stained with rabbit anti-PA28 ~i antiserum (
1:1000 dilution)
or anti-PA28a antiserum (1:200 dilution) followed by biotin-conjugated goat
anti-rabbit
IgG (1:100 dilution, Boehringer Mannheim, Montreal, QC) and streptavidin-
fluorescein.
The immunofluorescence of whole cells was examined and quantified with
confocal
microscopy as detailed before (Chen et al., 1997, J. Immunol. 159:905).
Proteinase assay
PBMC were cultured with or without PHA (2 ~zg/ml) and RAPA (lOnM). After 16h-
70h,
the cells were harvested and sonicated in 300 u1 PBS on ice for 40 sec. Twenty
micrograms of protein per sample from the cleared lysates were supplemented to
100 u1
with 0.1 M Tris buffer (pH 8.2). A proteasome-specific inhibitor lactacystin
(Omura et al.,
1991, supra; Fentenay et al., 1995, supra) was added at a final concentration
of IOnM in
some samples as indicated. The samples were incubated on ice for 15 min, and
fluorogenic
chymotrypsin substrate sLLVY-MCA was then added at a final concentration of
lOnM.
The 20S proteasome, which was prepared as previously described (Friguet et al,
1994, J.
Biol. Chem. 269:21639), was used as a positive control in place of cell
lysates. The
samples were incubated at 37°C for 15 min and the reaction was
terminated by adding 4 u1
2.5M HCI. The samples were then diluted to 2m1 with O.1M Tris pH8.2, and
measured for
their fluorescent intensity by a PTI fluorometer (Photo Technology
International, South
CA 02372316 2002-02-21
-53-
Brunswick, NJ). The excitation wavelength was 380 nm, and the emission
wavelength
440 nm.
EXAMPLE 5
The use of DPBA to treat allograft rejection in transplantation
Synthesis of DPBA
The applicant first synthesized DPBA (Fig. 26), and it had the expected
inhibitory effect to
the chymotrypsin-like activity of the 20S proteasome as shown in Fig. 27. The
ICso for the
inhibition of the chymotrypsin-like enzyme was about 20nM. DPBA also potently
inhibited proliferation of anti-CD3-stimulated T cells with ICSO of about 18
nM (Fig. 28),
which is consistent with DPBA's ICSO in enzyme inhibition. This showed that
DPBA, like
LAC, is effective in inhibiting T cell activation and proliferation in vitro.
Use of DPBA in mouse model of heart transplantation
The applicant then used DPBA in treating allograft rejection in a mouse model.
BALB/c
(H-2d) mice were used as donors and C57BL/6 (H-2b) was used as recipients.
Heterotropic
heart transplantation was performed as described in our previous publication
(Chen et al.,
1996, supra). As shown in Fig. 29, the control group had. mean survival rate
(MST) of 7.3
+ 0.5 (SD) days. When the recipients were administrated with straight
0.65mg/kg/day, i.p.
of DPBA for 16 days, the MST was more than 26.2 + 13 days. To mimic the
clinical
regimen of immunosuppressants, the applicant also tried a short-term high
dosage of
DPBA immediately after the transplantation (1 mg/kg/day, i.p. for 4 days from
day 1 to
day 4 post transplantation), followed by a low dosage (0.5 mg/kg/day, i.p.
from day 5 to
day 16). With this regimen, the MST is more than 22.8 + 9.8 days and has a
tendency of
being better than the first group. The mice appear healthy during or after the
drug
administration. These results for the first time show that a proteasome
inhibitor can be
used as an effective immunosuppressant in organ transplantation, and the
applicants have
CA 02372316 2002-02-21
-54-
proved that there exists a therapeutic dose window between the effective and
toxic dosages
of the proteasome inhibitor.
Treatment of ongoing rejection in the mouse heart transplantation model
Proteasome inhibitors when added at the late G1 phase can suppress
proliferation and even induce apoptosis of the activated T cells. This
suggests that the
inhibitors could treat ongoing rejection. This possibility was tested in mouse
heart
transplantation model. The recipients were given no immunosuppressants for 72
h after the
transplantation to allow the rejection response to proceed. Starting on day 3,
i.e. 72 h after
the operation, the mice were given DPBA at 1 mg/kg/day, i.p. for 4 days. As
shown in Fig.
30, the MST iS more than 13.2 + 1.78. The result suggests that the proteasome
inhibitor
will be useful in treating clinical rejection episodes, which are normally
diagnosed when
the rejection is ongoing. This new drug will be especially useful in patients
who are
resistant to commonly used immunosuppressants such as CyA, azathioprine, and
glucocorticoids.
The applicant has for the first time successfully used a proteasome
inhibitor to prevent allograft rejection. The proteasomes were thought to be
humble
"garbage collectors" to degrade cellular proteins in an unregulated way. The
applicant has
raised a novel concept and proved that the proteasome plays critical roles in
immune
regulation and the proteasome inhibitors can be used as novel
immunosuppressants in
organ_transplantation. The applicant have proved that there is a therapeutic
dose window
for the proteasome inhibitors in vivo, and the inhibitors are effective in
treating ongoing
graft rejection. Thus, the proteasome inhibitors, as represented by DPBA, are
a new class
of immunosuppressants. The usefulness of these class of immunosuppressants are
in
following three aspects: 1 ) They can be used alone, or in combination with
other
immunosuppressive drugs in alto or xeno organ transplantation; 2) They are
especially
useful in controlling clinical rejection episodes, which are normally
diagnosed when the T
cells are already activated, and are less responsive or resistant to
conventional
immunosuppressants; 3) They could be used in inducing long-term graft survival
by clonal
CA 02372316 2002-02-21
- 5~ -
deletion of alloantigen- or xenoantigen-specific T cells when administered
after the
activation of these cells; 4) By replacing the amino acid residues in the
DPBA, one could
generate proteasome inhibitors competitively inhibiting other protease
activities of the
proteasome, an some of them might have better therapeutic effects than the
model DPBA
used in this study. For example, one could replace the Phe and Leu in DPBA
with other
bulky hydrophobic amino acids to alter DPBA's inhibitory profile of the
chymotrypsin-
like activity of the proteasome; Lys and Arg can be used in the structure to
generate
inhibitors for the trypsin-like activity of the proteasome; Glu, branched
amino acids, and
small neutral amino acids could be used in the structure to generate
inhibitors for the
peptidylglutamyl peptide-hydrolyzing, branched chain amino-preferring, and
small neutral
amino acid-preferring activities, respectively.
The use of DPBA in organ transplantation - islet graft in streptozocin -
induced
diabetes in mice.
The islets from syngeneic mice (isograft control) restored normal glycemia in
diabetic
mice, and the effect lasted more than 60 days as expected. The allogeneic
islets were
rejected in about 10 days in untreated mice, and the mice became diabetic
after an initial
dip of their blood sugar level (allograft control). When the allogeneic islets
were
transplanted to diabetic recipients along with DPBA treatment, the graft
functionned
normally beyond 60 days, indicating that the graft rejection was inhibited.
This result
demonstrates that proteasome inhibitors as exemplified by DPBA can be used in
human
islet transplantation to prevent graft rejection. Fig. 31 shows that a
proteasome inhibitor
such as DPBA inhibits the glucose elevation consequent to islet rejection.
Conclusion
The proteasome inhibitors, represented hereinabove by LAC and
DPBA have shown a unique capacity to reverse an ongoing activity of blood
cells. This
CA 02372316 2002-02-21
-56-
reversal heretofore makes possible the treatment which selectively targets
activated blood
cells.
Although the present invention has been described hereinabove by way
of preferred embodiments thereof, it can be modified, without departing from
the spirit and
nature of the subject invention. Any such modification is under the scope of
this invention
as defined in the appended claims.
CA 02372316 2002-04-15
- 57 -
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: WU, Jiangping
WANG, Xin
(ii) TITLE OF INVENTION: The Use of Proteasome Inhibitors for
Treating Cancer, Inflammation, Autoimmune Disease, Graft
Rejection and Septic Shock
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Goudreau Gage Dubuc
(B) STREET: 3400 Stock Exchange Tower, PO Box 242 800
Place-Victoria
(C) CITY: Montreal
(D) STATE: Quebec
(E) COUNTRY: Canada
(F) ZIP: H4Z 1E9
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,372,316
(B) FILING DATE: 21-FEB-2002
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Britt, Katherine
(C) REFERENCE/DOCKET NUMBER: DH/12725.35
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (514) 397-7419
(B) TELEFAX: (514) 397-9382
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
GAAGAAGGGG GAGGATGA 18
CA 02372316 2002-04-15
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(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc ---- "Oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
AGCATTGCGG ATCTCCAT .Z$
